Compositions and methods for cancer expressing PDE3A or SLFN12

ABSTRACT

The present invention features improved methods of identifying patients having cancer (e.g., melanoma, adenocarcinoma, lung, cervical, liver or breast cancer) using biomarkers (e.g., PDE3A, SLFN12) that correlate with drug sensitivity and consequently treating a stratified patient population with an agent of the invention (e.g., DNMDP, zardaverine, and anagrelide).

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is the U.S. national phase application, pursuant to 35U.S.C. § 371, of PCT International Application Serial No.:PCT/US2016/046912, filed Aug. 12, 2016, designating the United Statesand published in English, which claims priority to and the benefit ofU.S. Provisional Application No. 62/204,875, filed Aug. 13, 2015, thedisclosure of which is incorporated herein by reference in its entirety.

STATEMENT OF RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSOREDRESEARCH

This invention was made with Government support under Grant No.3U54HG005032 awarded by the National Institutes of Health. TheGovernment has certain rights in the invention.

BACKGROUND OF THE INVENTION

Cancer kills over 550,000 people in the United States and over 8 millionpeople world-wide each year. New agents, including small molecules,molecules that impact tissue-specific growth requirements, andimmunomodulatory agents, have been shown to benefit a subset of patientswhose cancers have unique genomic mutations or other characteristics.Unfortunately, many cancer patients are still left without effectivetherapeutic options.

One approach to identify new anti-cancer agents is phenotypic screeningto discover novel small molecules displaying strong selectivity betweencancer cell lines, followed by chemogenomics to identify the cellfeatures associated with drug response. In the 1990s, Weinstein andcolleagues demonstrated that the cytotoxic profile of a compound can beused to identify cellular characteristics, such as gene-expressionprofiles and DNA copy number that correlate with drug sensitivity. Theability to identify the features of cancer cell lines that mediate theirresponse to small molecules has strongly increased in recent years withautomated high-throughput chemosensitivity testing of large panels ofcell lines coupled with comprehensive genomic and phenotypiccharacterization of the cell lines. Phenotypic observations ofsmall-molecule sensitivity can be linked to expression patterns orsomatic alterations, as in the case of SLFN11 expression in cancer celllines sensitive to irinotecan treatment, and an EWS-FLI1 rearrangementin cancer cell lines sensitive to PARP inhibitors, respectively.

Methods of characterizing malignancies at a molecular level are usefulfor stratifying patients, thereby quickly directing them to effectivetherapies. Improved methods for characterizing the responsiveness ofsubjects having cancer are urgently required.

SUMMARY OF THE INVENTION

As described below, the present invention features methods ofidentifying patients having a cancer (e.g., melanoma, adenocarcinoma,lung, cervical, liver, endometrium, lung, hematopoetic/lymphoid,ovarian, cervical, soft-tissue sarcoma, leiomyosarcoma, urinary tract,pancreas, thyroid, kidney, glioblastoma, or breast cancer) that issensitive to treatment with a phosphodiesterase 3A (PDE3A) modulator(e.g.,6-(4-(diethylamino)-3-nitrophenyl)-5-methyl-4,5-dihydropyridazin-3(2H)-one,zardaverine, and anagrelide) by detecting co-expression of PDE3A andSchlafen 12 (SLFN12) polynucleotides or polypeptides in a cancer cellderived from such patients.

In one embodiment, the present invention provides a method of killing orreducing the survival of a cancer cell selected as responsive to aphosphodiesterase 3A (PDE3A) modulator. The method includes the step ofcontacting the cell with a PDE3A modulator, where the cell was selectedas having an increase in PDE3A and/or Schlafen 12 (SLFN12) polypeptideor polynucleotide relative to a reference, thereby reducing the survivalof the cancer cell. In another embodiment, the present inventionprovides a method of reducing cancer cell proliferation in a subjectpre-selected as having a cancer that is responsive to a PDE3A modulator.The method comprises administering to the subject a PDE3A modulator,wherein the subject is pre-selected by detecting an increase in PDE3Aand/or SLFN12 polypeptide or polynucleotide levels relative to areference, thereby reducing cancer cell proliferation in the subject. Inone embodiment, the subject is pre-selected by detecting an increase inPDE3A and/or SLFN12 polypeptide or polynucleotide levels. In someembodiments, the PDE3A modulator is selected from the group consistingof6-(4-(diethylamino)-3-nitrophenyl)-5-methyl-4,5-dihydropyridazin-3(2H)-one(DNMDP), zardaverine, and anagrelide.

In another embodiment, the present invention provides a method ofidentifying a subject having a cancer responsive to PDE3A modulation.The method includes the step of detecting an increase in the level of aPDE3A and/or SLFN12 polypeptide or polynucleotide in a biological sampleof the subject relative to a reference, thereby identifying the subjectas responsive to PDE3A modulation. In one embodiment, an increase in thelevel of PDE3A and SFLN1 polypeptide or polynucleotide is detected.

In some embodiments, the increase in the level of PDE3A and/or SLFN12polypeptide is detected by a method selected from the group consistingof immunoblotting, mass spectrometry, and immunoprecipitation. In someother embodiments, the increase in the level of PDE3A, and/or SLFN12polynucleotide is detected by a method selected from the groupconsisting of quantitative PCR, Northern Blot, microarray, massspectrometry, and in situ hybridization. In some embodiments, theactivity of PDE3A is reduced. The PDE3A modulator may be administeredorally. The PDE3A modulator may be administered by intravenousinjection.

In some embodiments, the cancer cell is a melanoma, endometrium, lung,hematopoetic/lymphoid, ovarian, cervical, soft-tissue sarcoma,leiomyosarcoma, urinary tract, pancreas, thyroid, kidney, glioblastoma,or breast cancer. In some other embodiments, the cancer cell is not aB-cell proliferative type cancer. In some embodiments, the cancer cellis not multiple myeloma. In some embodiments, the biological sample is atissue sample.

In another aspect, the present invention provides a kit for identifyinga subject having cancer as responsive to PD3A modulation, the kitcomprising a capture reagent that binds PDE3A and/or a capture reagentthat binds SLFN12. In one embodiment, the kit comprises a capturereagent that binds PDE3A and a capture reagent that binds SLFN12.

In yet another aspect, the present invention provides a kit fordecreasing cancer cell proliferation in a subject pre-selected asresponsive to a PDE3A modulator, the kit comprising DNMDP, zardaverine,and/or anagrelide.

The invention provides methods for treating subjects having canceridentified as responsive to treatment with a PDE3A modulator bydetecting co-expression of PDE3A and/or Schlafen 12 (SLFN12)polynucleotides or polypeptides in the cancer. Compositions and articlesdefined by the invention were isolated or otherwise manufactured inconnection with the examples provided below. Other features andadvantages of the invention will be apparent from the detaileddescription, and from the claims.

Definitions

Unless defined otherwise, all technical and scientific terms used hereinhave the meaning commonly understood by a person skilled in the art towhich this invention belongs. The following references provide one ofskill with a general definition of many of the terms used in thisinvention: Singleton et al., Dictionary of Microbiology and MolecularBiology (2nd ed. 1994); The Cambridge Dictionary of Science andTechnology (Walker ed., 1988); The Glossary of Genetics, 5th Ed., R.Rieger et al. (eds.), Springer Verlag (1991); and Hale & Marham, TheHarper Collins Dictionary of Biology (1991). As used herein, thefollowing terms have the meanings ascribed to them below, unlessspecified otherwise.

By “Anagrelide” (IUPAC Name 6,7-dichloro-1,5-dihydroimidazo(2,1-b)quinazolin-2(3H)-one) is meant a small molecule phosphodiesteraseinhibitor having the following structure:

By “Cilostamide” (IUPAC NameN-cyclohexyl-N-methyl-4-[(2-oxo-1H-quinolin-6-yeoxy]butanamide) is meanta small molecule inhibitor having the following structure:

By “Cilostazol” (IUPAC Name6-[4-(1-cyclohexyl-1H-tetrazol-5-yl)butoxy]-3,4-dihydro-2(1H)-quinolinone)is meant a small molecule inhibitor having the following structure:

By “DNDMP” (IUPAC Name6-(4-(diethylamino)-3-nitrophenyl)-5-methyl-4,5-dihydropyridazin-3(2H)-one)is meant a small molecule inhibitor having the following structure:

By “Forskolin” (IUPAC Name(3R,4aR,5S,6S,6aS,10S,10aR,10bS)-6,10,10b-Trihydroxy-3,4a,7,7,10a-pentamethyl-1-oxo-3-vinyldodecahydro-1H-benzo[f]chromen-5-ylacetate)is meant a small molecule inhibitor having the following structure:

By “Levosimendan” (IUPAC Name(E)-2-cyano-1-methyl-3-(4-(4-methyl-6-oxo-1,4,5,6-tetrahydropyridazin-3-yl)phenyl)guanidine)is meant a small molecule inhibitor having the following structure:

By “Milrinone” (IUPAC Name2-methyl-6-oxo-1,6-dihydro-3,4′-bipyridine-5-carbonitrile) is meant asmall molecule inhibitor having the following structure:

By “Papaverine” (IUPAC Name1-(3,4-dimethoxybenzyl)-6,7-dimethoxyisoquinoline) is meant a smallmolecule inhibitor having the following structure:

By “Siguazodan” (IUPAC Name(E)-2-cyano-1-methyl-3-(4-(4-methyl-6-oxo-1,4,5,6-tetrahydropyridazin-3-yl)phenyl)guanidine)is meant a small molecule inhibitor having the following structure:

By “Sildenafil” (IUPAC Name1-[4-ethoxy-3-(6,7-dihydro-1-methyl-7-oxo-3-propyl-1H-pyrazolo[4,3-d]pyrimidin-5-yl)phenylsulfonyl]-4-methylpiperazine) is meant a small molecule inhibitor having thefollowing structure:

By “Trequinsin” (IUPAC Name9,10-dimethoxy-3-methyl-2-(2,4,6-trimethylphenyl)imino-6,7-dihydropyrimido[6,1-a]isoquinolin-4-one)is meant a small molecule inhibitor having the following structure:

By “Vardenifil” (IUPAC Name4-[2-ethoxy-5-(4-ethylpiperazin-1-yl)sulfonyl-phenyl]-9-methyl-7-propyl-3,5,6,8-tetrazabicyclo[4.3.0]nona-3,7,9-trien-2-one)is meant a small molecule inhibitor having the following structure:

By “Zardaverine (IUPAC Name3-[4-(Difluoromethoxy)-3-methoxyphenyl]-1H-pyridazin-6-one)” is meant asmall molecule inhibitor having the following structure:

In some other embodiments, any one of the compounds Cilostamide,Cilostazol, DNDMP, Levosimendan, Milrinone, Papaverine, Siguazodan,Sildenafil, Trequinsin, Vardenifil, and Zardaverine is a small moleculephosphodiesterase inhibitor. In another embodiment, forskolin may beused in a method of the invention.

By “PDE3A polypeptide” is meant a protein or fragment thereof having atleast 85% amino acid sequence identity to the sequence provided at NCBIRef No. NP_000912.3 that catalyzes the hydrolysis of cyclic adenosinemonophosphate (cAMP) and cyclic guanosine monophosphate (cGMP). Anexemplary human full-length PDE3A amino acid sequence is provided below:

(SEQ ID NO.: 3) MAVPGDAARVRDKPVHSGVSQAPTAGRDCHHRADPASPRDSGCRGCWGDLVLQPLRSSRKLSSALCAGSLSFLLALLVRLVRGEVGCDLEQCKEAAAAEEEEAAPGAEGGVFPGPRGGAPGGGARLSPWLQPSALLFSLLCAFFWMGLYLLRAGVRLPLAVALLAACCGGEALVQIGLGVGEDHLLSLPAAGVVLSCLAAATWLVLRLRLGVLMIALTSAVRTVSLISLERFKVAWRPYLAYLAGVLGILLARYVEQILPQSAEAAPREHLGSQLIAGTKEDIPVFKRRRRSSSVVSAEMSGCSSKSHRRTSLPCIPREQLMGHSEWDHKRGPRGSQSSGTSITVDIAVMGEAHGLITDLLADPSLPPNVCTSLRAVSNLLSTQLTFQAIHKPRVNPVTSLSENYTCSDSEESSEKDKLAIPKRLRRSLPPGLLRRVSSTWTTTTSATGLPTLEPAPVRRDRSTSIKLQEAPSSSPDSWNNPVMMTLTKSRSFTSSYAISAANHVKAKKQSRPGALAKISPLSSPCSSPLQGTPASSLVSKISAVQFPESADTTAKQSLGSHRALTYTQSAPDLSPQILTPPVICSSCGRPYSQGNPADEPLERSGVATRTPSRTDDTAQVTSDYETNNNSDSSDIVQNEDETECLREPLRKASACSTYAPETMMFLDKPILAPEPLVMDNLDSIMEQLNTWNFPIFDLVENIGRKCGRILSQVSYRLFEDMGLFEAFKIPIREFMNYFHALEIGYRDIPYHNRIHATDVLHAVWYLTTQPIPGLSTVINDHGSTSDSDSDSGFTHGHMGYVFSKTYNVTDDKYGCLSGNIPALELMALYVAAAMHDYDHPGRTNAFLVATSAPQAVLYNDRSVLENHHAAAAWNLFMSRPEYNFLINLDHVEFKHFRFLVIEAILATDLKKHFDFVAKFNGKVNDDVGIDWTNENDRLLVCQMCIKLADINGPAKCKELHLQWTDGIVNEFYEQGDEEASLGLPISPFMDRSAPQLANLQESFISHIVGPLCNSYDSAGLMPGKWVEDSDESGDTDDPEEEEEEAPAPNEEETCENNESPKKKTFKRRKIYOQITQHLLQNHKMWKKVIEEEQRLAGIENQSLDQTPQSHSSEQIQAIKEEEEEKGKPRGEEIPTQKPDQThree PDE3A isoforms are known: PDE3A1, PDE3A2, and PDE3A3. PDE3A1comprises amino acids 146-1141, PDE3A2 isoform 2 comprises amino acids299-1141, and PDE3A3 comprises amino acids 483-1141 of the full-lengthPDE3A amino acid sequence.

By “PDE3A polynucleotide” is meant any nucleic acid molecule, includingDNA and RNA, encoding a PDE3A polypeptide or fragment thereof. Anexemplary PDE3A nucleic acid sequence is provided at NCBI Ref:NM_000921.4:

(SEQ ID NO.: 4) 1 gggggccact gggaattcag tgaagagggc accctataccatggcagtgc ccggcgacgc 61 tgcacgagtc agggacaagc ccgtccacag tggggtgagtcaagccccca cggcgggccg 121 ggactgccac catcgtgcgg accccgcatc gccgcgggactcgggctgcc gtggctgctg 181 gggagacctg gtgctgcagc cgctccggag ctctcggaaactttcctccg cgctgtgcgc 241 gggctccctg tcctttctgc tggcgctgct ggtgaggctggtccgcgggg aggtcggctg 301 tgacctggag cagtgtaagg aggcggcggc ggcggaggaggaggaagcag ccccgggagc 361 agaagggggc gtcttcccgg ggcctcgggg aggtgctcccgggggcggtg cgcggctcag 421 cccctggctg cagccctcgg cgctgctctt cagtctcctgtgtgccttct tctggatggg 481 cttgtacctc ctgcgcgccg gggtgcgcct gcctctggctgtcgcgctgc tggccgcctg 541 ctgcgggggg gaagcgctcg tccagattgg gctgggcgtcggggaggatc acttactctc 601 actccccgcc gcgggggtgg tgctcagctg cttggccgccgcgacatggc tggtgctgag 661 gctgaggctg ggcgtcctca tgatcgcctt gactagcgcggtcaggaccg tgtccctcat 721 ttccttagag aggttcaagg tcgcctggag accttacctggcgtacctgg ccggcgtgct 781 ggggatcctc ttggccaggt acgtggaaca aatcttgccgcagtccgcgg aggcggctcc 841 aagggagcat ttggggtccc agctgattgc tgggaccaaggaagatatcc cggtgtttaa 901 gaggaggagg cggtccagct ccgtcgtgtc cgccgagatgtccggctgca gcagcaagtc 961 ccatcggagg acctccctgc cctgtatacc gagggaacagctcatggggc attcagaatg 1021 ggaccacaaa cgagggccaa gaggatcaca gtcttcaggaaccagtatta ctgtggacat 1081 cgccgtcatg ggcgaggccc acggcctcat taccgacctcctggcagacc cttctcttcc 1141 accaaacgtg tgcacatcct tgagagccgt gagcaacttgctcagcacac agctcacctt 1201 ccaggccatt cacaagccca gagtgaatcc cgtcacttcgctcagtgaaa actatacctg 1261 ttctgactct gaagagagct ctgaaaaaga caagcttgctattccaaagc gcctgagaag 1321 gagtttgcct cctggcttgt tgagacgagt ttcttccacttggaccacca ccacctcggc 1381 cacaggtcta cccaccttgg agcctgcacc agtacggagagaccgcagca ccagcatcaa 1441 actgcaggaa gcaccttcat ccagtcctga ttcttggaataatccagtga tgatgaccct 1501 caccaaaagc agatccttta cttcatccta tgctatttctgcagctaacc atgtaaaggc 1561 taaaaagcaa agtcgaccag gtgccctcgc taaaatttcacctctttcat cgccctgctc 1621 ctcacctctc caagggactc ctgccagcag cctggtcagcaaaatttctg cagtgcagtt 1681 tccagaatct gctgacacaa ctgccaaaca aagcctaggttctcacaggg ccttaactta 1741 cactcagagt gccccagacc tatcccctca aatcctgactccacctgtta tatgtagcag 1801 ctgtggcaga ccatattccc aagggaatcc tgctgatgagcccctggaga gaagtggggt 1861 agccactcgg acaccaagta gaacagatga cactgctcaagttacctctg attatgaaac 1921 caataacaac agtgacagca gtgacattgt acagaatgaagatgaaacag agtgcctgag 1981 agagcctctg aggaaagcat cggcttgcag cacctatgctcctgagacca tgatgtttct 2041 ggacaaacca attcttgctc ccgaacctct tgtcatggataacctggact caattatgga 2101 gcagctaaat acttggaatt ttccaatttt tgatttagtggaaaatatag gaagaaaatg 2161 tggccgtatt cttagtcagg tatcttacag actttttgaagacatgggcc tctttgaagc 2221 ttttaaaatt ccaattaggg aatttatgaa ttattttcatgctttggaga ttggatatag 2281 ggatattcct tatcataaca gaatccatgc cactgatgttttacatgctg tttggtatct 2341 tactacacag cctattccag gcctctcaac tgtgattaatgatcatggtt caaccagtga 2401 ttcagattct gacagtggat ttacacatgg acatatgggatatgtattct caaaaacgta 2461 taatgtgaca gatgataaat acggatgtct gtctgggaatatccctgcct tggagttgat 2521 ggcgctgtat gtggctgcag ccatgcacga ttatgatcatccaggaagga ctaatgcttt 2581 cctggttgca actagtgctc ctcaggcggt gctatataacgatcgttcag ttttggagaa 2641 tcatcacgca gctgctgcat ggaatctttt catgtcccggccagagtata acttcttaat 2701 taaccttgac catgtggaat ttaagcattt ccgtttccttgtcattgaag caattttggc 2761 cactgacctg aagaaacact ttgacttcgt agccaaatttaatggcaagg taaatgatga 2821 tgttggaata gattggacca atgaaaatga tcgtctactggtttgtcaaa tgtgtataaa 2881 gttggctgat atcaatggtc cagctaaatg taaagaactccatcttcagt ggacagatgg 2941 tattgtcaat gaattttatg aacagggtga tgaagaggccagccttggat tacccataag 3001 ccccttcatg gatcgttctg ctcctcagct ggccaaccttcaggaatcct tcatctctca 3061 cattgtgggg cctctgtgca actcctatga ttcagcaggactaatgcctg gaaaatgggt 3121 ggaagacagc gatgagtcag gagatactga tgacccagaagaagaggagg aagaagcacc 3181 agcaccaaat gaagaggaaa cctgtgaaaa taatgaatctccaaaaaaga agactttcaa 3241 aaggagaaaa atctactgcc aaataactca gcacctcttacagaaccaca agatgtggaa 3301 gaaagtcatt gaagaggagc aacggttggc aggcatagaaaatcaatccc tggaccagac 3361 ccctcagtcg cactcttcag aacagatcca ggctatcaaggaagaagaag aagagaaagg 3421 gaaaccaaga ggcgaggaga taccaaccca aaagccagaccagtgacaat ggatagaatg 3481 ggctgtgttt ccaaacagat tgacttgtca aagactctcttcaagccagc acaacattta 3541 gacacaacac tgtagaaatt tgagatgggc aaatggctattgcattttgg gattcttcgc 3601 attttgtgtg tatattttta cagtgaggta cattgttaaaaactttttgc tcaaagaagc 3661 tttcacattg caacaccagc ttctaaggat tttttaaggagggaatatat atgtgtgtgt 3721 gtatataagc tcccacatag atacatgtaa aacatattcacacccatgca cgcacacaca 3781 tacacactga aggccacgat tgctggctcc acaatttagtaacatttata ttaagatata 3841 tatatagtgg tcactgtgat ataataaatc ataaaggaaaccaaatcaca aaggagatgg 3901 tgtggcttag caaggaaaca gtgcaggaaa tgtaggttaccaactaagca gcttttgctc 3961 ttagtactga gggatgaaag ttccagagca ttatttgaattctgatacat cctgccaaca 4021 ctgtgtgtgt gtgtgtgtgt gtgtgtgtgt gtgtgtgtgtgtgtgaaaga gagacagaag 4081 ggaatggttt gagagggtgc ttgtgtgcat gtgtgtgcatatgtaaagag atttttgtgg 4141 tttaagtaac tcagaatagc tgtagcaaat gactgaatacatgtgaacaa acagaaggaa 4201 gttcactctg gagtgtcttt gggaggcagc cattccaaatgccctcctcc atttagcttc 4261 aataaagggc cttttgctga tggagggcac tcaagggctgggtgagaggg ccacgtgttt 4321 ggtattacat tactgctatg caccacttga aggagctctatcaccagcct caaacccgaa 4381 agactgaggc attttccagt ctacttgcct aatgaatgtataggaactgt ctatgagtat 4441 ggatgtcact caactaagat caaatcacca tttaaggggatggcattctt tatacctaaa 4501 cacctaagag ctgaagtcag gtcttttaat caggttagaattctaaatga tgccagagaa 4561 ggcttgggaa attgtacttc agcgtgatag cctgtgtcttcttaatttgc tgcaaaatat 4621 gtggtagaga aagaaaagga aacagaaaaa tcactctgggttatatagca agagatgaag 4681 gagaatattt caacacaggg tttttgtgtt gacataggaaaagcctgatt cttggcaact 4741 gttgtagttt gtctttcagg ggtgaaggtc ccactgacaacccctgttgt ggtgttccac 4801 acgctgtttg ttggggtagc ttccatcggc agtctggcccattgtcagtc atgcttcttc 4861 tggccgggga gattatagag agattgtttg aagattgggttattattgaa agtctttttt 4921 tttgtttgtt ttgttttggt ttgtttgttt atctacacttgtttatgctg tgagccaaac 4981 ctctatttaa aaagttgata ctcactttca atattttatttcatattatt atatatgtca 5041 tgatagttat cttgatgtaa atatgaagat ttttttgtttctgtagatag taaactcttt 5101 ttttaaaaaa ggaaaaggga aacattttta taaagttatattttaatcac catttttata 5161 cattgtagtt ctctccaagc ccagtaagag aatgatgattcatttgcatg gaggtcgatg 5221 gacaaccaat catctacctt ttctaattta aatgataatctgatatagtt ttattgccag 5281 ttaaatgagg atgctgcaaa gcatgttttt tcactagtaacttttgctaa ctgaatgaat 5341 tctgggtcca tatctcccag atgaaaaact gttaaccaataccatatttt atagttggtg 5401 tccatttctt tccaacactg tttgttatga ttcttccttgagtacttata tacagacctg 5461 ctcattatct aaacaatctt accttctaag taaaccttgattgtgatttc cagtttttat 5521 tttctctgac gtagtagaaa ggaatgttta cattaaaaatacttttgttt ctcataaatg 5581 gatattgtac tccccccttt caaagcatta ttttacaataattcatggca ttttaaaaaa 5641 taaggcaaag ataatacgac aaaaaatata catggtttcaaggcaaattc tccaataagt 5701 tggaaaatgt aaaaaggatc aagtggatgc agcctctacctaaataatta aaatatattt 5761 cagtatattt ctgaattaac accaggtctt cattatttagaacttactaa attgttttca 5821 ttttcttagt tttacctgtg tatctccatg tttgcaaaaattactataag tcaaattttg 5881 ccagtgaatt taactatttt tctttccttg caattaaggggaaaaaagca tttatcttat 5941 cttctcatac cccttgcatc taagtactta gcaaagtcaatattttccca ttttccaaat 6001 gcgtccatct ctaacataaa tattaattga acatagagctatgtttggag tgagtggact 6061 ggcaggacag ttggaagtcc atcacagtct attgacagtttcatcaaagc tgtatagtcc 6121 aactagtggg gcagcttggc tactatggtg gaagtctcagcaaactgcct ggttttgttt 6181 gtttgttttg ttttaaggta caggaaataa gaggaataatagtggccaaa gcaattagaa 6241 catcttcatt ccagaactgt gttcagcaat ccaggcagattgatacattt ttctttaaaa 6301 ataaattgct attacagcta gacgtcaatt gggataaataaagggatgaa gatccactaa 6361 gtttgtgact ttcatacaca cccagtacat ctcaaaggatgctaagggac attttctgcc 6421 agtagagttc tccccctttt tggtgacagc aatattattatgttcacatc taactccaga 6481 gcttacttcc tgtggtgcca atgtatttgt tgcaatttactacattttta tatgagccta 6541 tttataggtg ccattaaact caggtctttc aaatgaaagagtttctagcc cacttaggga 6601 aaaagataat tgtttagaaa accataaaat caatggtaggaaaagttgga actggttacc 6661 tggatgccat ggttctctgt taaataaagt aagagaccaggtgtattctg agtgtcatca 6721 gtgttatttt cagcatgcta ataaatgtct ttccggttatatatctatct aaattaacct 6781 ttaaaatatt ggtttccttg ataaaagcac cacttttgcttttgttagct gtaatatttt 6841 ttgtcattta gataagacct ggtttggctc tcaataaaagatgaagacag tagctctgta 6901 cagggatata tctatattag tcttcatctg atgaatgaagaaattttctc atattatgtt 6961 caagaaagta tttacttcct aaaaatagaa ttcccgattctgtctatttt ggttgaatac 7021 cagaacaaat ctttccgttg caatcccagt aaaacgaaagaaaaggaata tcttacagac 7081 tgttcatatt agatgtatgt agactgttaa tttgcaatttccccatattt cctgcctatc 7141 ttacccagat aactttcttt gaaggtaaaa gctgtgcaaaaggcatgaga ctcaggccta 7201 ctctttgttt aaatgatgga aaaatataaa ttattttctaagtaataaaa gtataaaaat 7261 tatcattata aataaagtct aaagtttgaa attattaatttaaaaaaaaa aaaaaaaaa

By “Schlafen 12 (SLFN12) polypeptide” is meant a protein or fragmentthereof having at least 85% amino acid sequence identity to the sequenceprovided at NCBI Ref No. NP_060512.3 that interacts with PDE3A whenbound to anagrelide, zardaverine or DNMDP and related compounds. Anexemplary human SLFN12 amino acid sequence is provided below:

(SEQ ID NO.: 5) MNISVDLETNYAELVLDVGRVTLGENSRKKMKDCKLRKKQNESVSRAMCALLNSGGGVIKAEIENEDYSYTKDGIGLDLENSFSNILLFVPEYLDFMQNGNYFLIFVKSWSLNTSGLRITTLSSNLYKRDITSAKVMNATAALEFLKDMKKTRGRLYLRPELLAKRPCVDIQEENNMKALAGVFFDRTELDRKEKLTFTESTHVEIKNESTEKLLQRIKEILPQYVSAFANTDGGYLFIGLNEDKEIIGFKAEMSDLDDLEREIEKSIRKMPVHHFCMEKKKINYSCKFLGVYDKGSLCGYVCALRVERFCCAVFAKEPDSWHVKDNRVMQLTRKEWIQFMVEAEPKFSSSYEEVISQINTSLPAPHSWPLLEWQRQRHHCPGLSGRITYTPENLCRKLFLQHEGLKQLICEEMDSVRKGSLIFSRSWSVDLGLQENHKVLCDALLISQDSPPVLYTFHMVQDEEFKGYSTQTALTLKQKLAKIGGYTKKVCVMTKIFYLSPEGMTSCQYDLRSQVIYPESYYFTRRKYLLKALFKALKRLKSLRDQFSFAENLYQIIGIDCFQKNDKKMFKSCRRLT

By “Schlafen 12 (SLFN12) polynucleotide” is meant any nucleic acidmolecule, including DNA and RNA, encoding a SLFN12 polypeptide orfragment thereof. An exemplary SLFN12 nucleic acid sequence is providedat NCBI Ref: NM_018042.4:

(SEQ ID NO.: 6) 1 tttgtaactt cacttcagcc tcccattgat cgctttctgcaaccattcag actgatctcg 61 ggctcctatt tcatttacat tgtgtgcaca ccaagtaaccagtgggaaaa ctttagaggg 121 tacttaaacc ccagaaaatt ctgaaaccgg gctcttgagccgctatcctc gggcctgctc 181 ccaccctgtg gagtgcactt tcgttttcaa taaatctctgcttttgttgc ttcattcttt 241 ccttgctttg tttgtgtgtt tgtccagttc tttgttcaacacgccaagaa cctggacact 301 cttcactggt aacatatttt ggcaagccaa ccaggagaaaagaatttctg cttggacact 361 gcatagctgc tgggaaaatg aacatcagtg ttgatttggaaacgaattat gccgagttgg 421 ttctagatgt gggaagagtc actcttggag agaacagtaggaaaaaaatg aaggattgta 481 aactgagaaa aaagcagaat gaaagtgtct cacgagctatgtgtgctctg ctcaattctg 541 gagggggagt gatcaaggct gaaattgaga atgaagactatagttataca aaagatggaa 601 taggactaga tttggaaaat tcttttagta acattctgttatttgttcct gagtacttag 661 acttcatgca gaatggtaac tactttctga tttttgtgaagtcatggagc ttgaacacct 721 ctggtctgcg gattaccacc ttgagctcca atttgtacaaaagagatata acatctgcaa 781 aagtcatgaa tgccactgct gcactggagt tcctcaaagacatgaaaaag actagaggga 841 gattgtattt aagaccagaa ttgctggcaa agaggccctgtgttgatata caagaagaaa 901 ataacatgaa ggccttggcc ggggtttttt ttgatagaacagaacttgat cggaaagaaa 961 aattgacctt tactgaatcc acacatgttg aaattaaaaacttctcgaca gaaaagttgt 1021 tacaacgaat taaagagatt ctccctcaat atgtttctgcatttgcaaat actgatggag 1081 gatatttgtt cattggttta aatgaagata aagaaataattggctttaaa gcagagatga 1141 gtgacctcga tgacttagaa agagaaatcg aaaagtccattaggaagatg cctgtgcatc 1201 acttctgtat ggagaagaag aagataaatt attcatgcaaattccttgga gtatatgata 1261 aaggaagtct ttgtggatat gtctgtgcac tcagagtggagcgcttctgc tgtgcagtgt 1321 ttgctaaaga gcctgattcc tggcatgtga aagataaccgtgtgatgcag ttgaccagga 1381 aggaatggat ccagttcatg gtggaggctg aaccaaaattttccagttca tatgaagagg 1441 tgatctctca aataaatacg tcattacctg ctccccacagttggcctctt ttggaatggc 1501 aacggcagag acatcactgt ccagggctat caggaaggataacgtatact ccagaaaacc 1561 tttgcagaaa actgttctta caacatgaag gacttaagcaattaatatgt gaagaaatgg 1621 actctgtcag aaagggctca ctgatcttct ctaggagctggtctgtggat ctgggcttgc 1681 aagagaacca caaagtcctc tgtgatgctc ttctgatttcccaggacagt cctccagtcc 1741 tatacacctt ccacatggta caggatgagg agtttaaaggctattctaca caaactgccc 1801 taaccttaaa gcagaagctg gcaaaaattg gtggttacactaaaaaagtg tgtgtcatga 1861 caaagatctt ctacttgagc cctgaaggca tgacaagctgccagtatgat ttaaggtcgc 1921 aagtaattta ccctgaatcc tactatttta caagaaggaaatacttgctg aaagcccttt 1981 ttaaagcctt aaagagactc aagtctctga gagaccagttttcctttgca gaaaatctat 2041 accagataat cggtatagat tgctttcaga agaatgataaaaagatgttt aaatcttgtc 2101 gaaggctcac ctgatggaaa atggactggg ctactgagatatttttcatt atatatttga 2161 taacattctc taattctgtg aaaatatttc tttgaaaactttgcaagtta agcaacttaa 2221 tgtgatgttg gataattggg ttttgtctat tttcacttctccctaaataa tcttcacaga 2281 tattgtttga gggatattag gaaaattaat ttgttaactcgtctgtgcac agtattattt 2341 actctgtctg tagttcctga ataaattttc ttccatgcttgaactgggaa aattgcaaca 2401 cttttattct taatgacaac agtgaaaatc tcccagcatatacctagaaa acaattataa 2461 cttacaaaag attatccttg atgaaactca gaatttccacagtgggaatg aataagaagg 2521 caaaactcat

In some aspects, the compound is an isomer. “Isomers” are differentcompounds that have the same molecular formula. “Stereoisomers” areisomers that differ only in the way the atoms are arranged in space. Asused herein, the term “isomer” includes any and all geometric isomersand stereoisomers. For example, “isomers” include geometric double bondcis- and trans-isomers, also termed E- and Z-isomers; R- andS-enantiomers; diastereomers, (d)-isomers and (l)-isomers, racemicmixtures thereof; and other mixtures thereof, as falling within thescope of this invention.

Geometric isomers can be represented by the symbol

which denotes a bond that can be a single, double or triple bond asdescribed herein. Provided herein are various geometric isomers andmixtures thereof resulting from the arrangement of substituents around acarbon-carbon double bond or arrangement of substituents around acarbocyclic ring. Substituents around a carbon-carbon double bond aredesignated as being in the “Z” or “E” configuration wherein the terms“Z” and “E” are used in accordance with IUPAC standards. Unlessotherwise specified, structures depicting double bonds encompass boththe “E” and “Z” isomers.

Substituents around a carbon-carbon double bond alternatively can bereferred to as “cis” or “trans,” where “cis” represents substituents onthe same side of the double bond and “trans” represents substituents onopposite sides of the double bond. The arrangement of substituentsaround a carbocyclic ring can also be designated as “cis” or “trans.”The term “cis” represents substituents on the same side of the plane ofthe ring, and the term “trans” represents substituents on opposite sidesof the plane of the ring. Mixtures of compounds wherein the substituentsare disposed on both the same and opposite sides of plane of the ringare designated “cis/trans.”

The term “enantiomers” refers to a pair of stereoisomers that arenon-superimposable mirror images of each other. An atom having anasymmetric set of substituents can give rise to an enantiomer. A mixtureof a pair of enantiomers in any proportion can be known as a “racemic”mixture. The term “(±)” is used to designate a racemic mixture whereappropriate. “Diastereoisomers” are stereoisomers that have at least twoasymmetric atoms, but which are not mirror-images of each other. Theabsolute stereochemistry is specified according to theCahn-Ingold-Prelog R-S system. When a compound is an enantiomer, thestereochemistry at each chiral carbon can be specified by either R or S.Resolved compounds whose absolute configuration is unknown can bedesignated (+) or (−) depending on the direction (dextro- orlevorotatory) which they rotate plane polarized light at the wavelengthof the sodium D line. Certain of the compounds described herein containone or more asymmetric centers and can thus give rise to enantiomers,diastereomers, and other stereoisomeric forms that can be defined, interms of absolute stereochemistry at each asymmetric atom, as (R)- or(S)-. The present chemical entities, pharmaceutical compositions andmethods are meant to include all such possible isomers, includingracemic mixtures, optically substantially pure forms and intermediatemixtures.

Optically active (R)- and (S)-isomers can be prepared, for example,using chiral synthons or chiral reagents, or resolved using conventionaltechniques. Enantiomers can be isolated from racemic mixtures by anymethod known to those skilled in the art, including chiral high pressureliquid chromatography (HPLC), the formation and crystallization ofchiral salts, or prepared by asymmetric syntheses.

Optical isomers can be obtained by resolution of the racemic mixturesaccording to conventional processes, e.g., by formation ofdiastereoisomeric salts, by treatment with an optically active acid orbase. Examples of appropriate acids are tartaric, diacetyltartaric,dibenzoyltartaric, ditoluoyltartaric, and camphorsulfonic acid. Theseparation of the mixture of diastereoisomers by crystallizationfollowed by liberation of the optically active bases from these saltsaffords separation of the isomers. Another method involves synthesis ofcovalent diastereoisomeric molecules by reacting disclosed compoundswith an optically pure acid in an activated form or an optically pureisocyanate. The synthesized diastereoisomers can be separated byconventional means such as chromatography, distillation, crystallizationor sublimation, and then hydrolyzed to deliver the enantiomericallyenriched compound. Optically active compounds can also be obtained byusing active starting materials. In some embodiments, these isomers canbe in the form of a free acid, a free base, an ester or a salt.

In certain embodiments, the compound of the invention can be a tautomer.As used herein, the term “tautomer” is a type of isomer that includestwo or more interconvertible compounds resulting from at least oneformal migration of a hydrogen atom and at least one change in valency(e.g., a single bond to a double bond, a triple bond to a single bond,or vice versa). “Tautomerization” includes prototropic or proton-shifttautomerization, which is considered a subset of acid-base chemistry.“Prototropic tautomerization” or “proton-shift tautomerization” involvesthe migration of a proton accompanied by changes in bond order. Theexact ratio of the tautomers depends on several factors, includingtemperature, solvent, and pH. Where tautomerization is possible (e.g.,in solution), a chemical equilibrium of tautomers can be reached.Tautomerizations (i.e., the reaction providing a tautomeric pair) can becatalyzed by acid or base, or can occur without the action or presenceof an external agent. Exemplary tautomerizations include, but are notlimited to, keto-to-enol; amide-to-imide; lactam-to-lactim;enamine-to-imine; and enamine-to-(a different) enamine tautomerizations.A specific example of keto-enol tautomerization is the interconversionof pentane-2,4-dione and 4-hydroxypent-3-en-2-one tautomers. Anotherexample of tautomerization is phenol-keto tautomerization. A specificexample of phenol-keto tautomerization is the interconversion ofpyridin-4-ol and pyridin-4(1H)-one tautomers.

All chiral, diastereomeric, racemic, and geometric isomeric forms of astructure are intended, unless specific stereochemistry or isomeric formis specifically indicated. All processes used to prepare compounds ofthe present invention and intermediates made therein are considered tobe part of the present invention. All tautomers of shown or describedcompounds are also considered to be part of the present invention.

By “agent” is meant any small molecule chemical compound, antibody,nucleic acid molecule, or polypeptide, or fragments thereof.

By “ameliorate” is meant decrease, suppress, attenuate, diminish,arrest, or stabilize the development or progression of a disease.

By “alteration” is meant a change (increase or decrease) in theexpression levels or activity of a gene or polypeptide as detected bystandard art known methods such as those described herein. As usedherein, an alteration includes an about 10% change in expression levels,preferably an about 25% change, more preferably an about 40% change, andmost preferably an about 50% or greater change in expression levels.”

By “analog” is meant a molecule that is not identical, but has analogousfunctional or structural features. For example, a polypeptide analogretains the biological activity of a corresponding naturally-occurringpolypeptide, while having certain biochemical modifications that enhancethe analog's function relative to a naturally occurring polypeptide.Such biochemical modifications could increase the analog's proteaseresistance, membrane permeability, or half-life, without altering, forexample, ligand binding. An analog may include an unnatural amino acid.

In this disclosure, “comprises,” “comprising,” “containing” and “having”and the like can have the meaning ascribed to them in U.S. Patent lawand can mean “includes,” “including,” and the like; “consistingessentially of” or “consists essentially” likewise has the meaningascribed in U.S. patent law and the term is open-ended, allowing for thepresence of more than that which is recited so long as basic or novelcharacteristics of that which is recited is not changed by the presenceof more than that which is recited, but excludes prior art embodiments.

“Detect” refers to identifying the presence, absence or amount of theanalyte to be detected. In particular embodiments, the analyte is aPDE3A or SLFN12 polypeptide.

By “disease” is meant any condition or disorder that damages orinterferes with the normal function of a cell, tissue, or organ.Examples of diseases include melanoma, adenocarcinoma, lung cancer,cervical cancer, liver cancer and breast cancer.

By “effective amount” is meant the amount of a compound described hereinrequired to ameliorate the symptoms of a disease relative to anuntreated patient. The effective amount of active compound(s) used topractice the present invention for therapeutic treatment of a diseasevaries depending upon the manner of administration, the age, bodyweight, and general health of the subject. Ultimately, the attendingphysician or veterinarian will decide the appropriate amount and dosageregimen. Such amount is referred to as an “effective” amount. In oneembodiment, the compound is DNMDP, zardaverine, or anagrelide.

The invention provides a number of targets that are useful for thedevelopment of highly specific drugs to treat or a disordercharacterized by the methods delineated herein. In addition, the methodsof the invention provide a facile means to identify therapies that aresafe for use in subjects. In addition, the methods of the inventionprovide a route for analyzing virtually any number of compounds foreffects on a disease described herein with high-volume throughput, highsensitivity, and low complexity.

By “fragment” is meant a portion of a polypeptide or nucleic acidmolecule. This portion contains, preferably, at least about 10%, about20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%,or about 90% of the entire length of the reference nucleic acid moleculeor polypeptide. A fragment may contain about 10, about 20, about 30,about 40, about 50, about 60, about 70, about 80, about 90, about 100,about 200, about 300, about 400, about 500, about 600, about 700, about800, about 900, or about 1000 nucleotides or amino acids.

“Hybridization” means hydrogen bonding, which may be Watson-Crick,Hoogsteen or reversed Hoogsteen hydrogen bonding, between complementarynucleobases. For example, adenine and thymine are complementarynucleobases that pair through the formation of hydrogen bonds.

By “marker” or “biomarker” is meant any protein or polynucleotide havingan alteration in expression level or activity (e.g., at the protein ormRNA level) that is associated with a disease or disorder. In particularembodiments, a marker of the invention is PDE3A or SLFN12.

By “modulator” is meant any agent that binds to a polypeptide and altersa biological function or activity of the polypeptide. A modulatorincludes, without limitation, agents that reduce or eliminate abiological function or activity of a polypeptide (e.g., an “inhibitor”).For example, a modulator may inhibit a catalytic activity of apolypeptide. A modulator includes, without limitation, agents thatincrease or decrease binding of a polypeptide to another agent. Forexample, a modulator may promote binding of a polypeptide to anotherpolypeptide. In some embodiments, a modulator of PDE3A polypeptide isDNMDP. In some other embodiments, the modulator of PDE3A polypeptide isanagrelide or zardaverine.

By “reference” is meant a standard or control condition.

Nucleic acid molecules useful in the methods of the invention includeany nucleic acid molecule that encodes a polypeptide of the invention ora fragment thereof. Such nucleic acid molecules need not be 100%identical with an endogenous nucleic acid sequence, but will typicallyexhibit substantial identity. Polynucleotides having “substantialidentity” to an endogenous sequence are typically capable of hybridizingwith at least one strand of a double-stranded nucleic acid molecule.Nucleic acid molecules useful in the methods of the invention includeany nucleic acid molecule that encodes a polypeptide of the invention ora fragment thereof. Such nucleic acid molecules need not be 100%identical with an endogenous nucleic acid sequence, but will typicallyexhibit substantial identity. Polynucleotides having “substantialidentity” to an endogenous sequence are typically capable of hybridizingwith at least one strand of a double-stranded nucleic acid molecule. By“hybridize” is meant pair to form a double-stranded molecule betweencomplementary polynucleotide sequences (e.g., a gene described herein),or portions thereof, under various conditions of stringency. (See, e.g.,Wahl, G. M. and S. L. Berger (1987) Methods Enzymol. 152:399; Kimmel, A.R. (1987) Methods Enzymol. 152:507).

For example, stringent salt concentration will ordinarily be less thanabout 750 mM NaCl and 75 mM trisodium citrate, preferably less thanabout 500 mM NaCl and 50 mM trisodium citrate, and more preferably lessthan about 250 mM NaCl and 25 mM trisodium citrate. Low stringencyhybridization can be obtained in the absence of organic solvent, e.g.,formamide, while high stringency hybridization can be obtained in thepresence of at least about 35% formamide, and more preferably at leastabout 50% formamide. Stringent temperature conditions will ordinarilyinclude temperatures of at least about 30° C., more preferably of atleast about 37° C., and most preferably of at least about 42° C. Varyingadditional parameters, such as hybridization time, the concentration ofdetergent, e.g., sodium dodecyl sulfate (SDS), and the inclusion orexclusion of carrier DNA, are well known to those skilled in the art.Various levels of stringency are accomplished by combining these variousconditions as needed. In a preferred: embodiment, hybridization willoccur at 30° C. in 750 mM NaCl, 75 mM trisodium citrate, and 1% SDS. Ina more preferred embodiment, hybridization will occur at 37° C. in 500mM NaCl, 50 mM trisodium citrate, 1% SDS, 35% formamide, and 100 μg/mldenatured salmon sperm DNA (ssDNA). In a most preferred embodiment,hybridization will occur at 42° C. in 250 mM NaCl, 25 mM trisodiumcitrate, 1% SDS, 50% formamide, and 200 μg/ml ssDNA. Useful variationson these conditions will be readily apparent to those skilled in theart.

For most applications, washing steps that follow hybridization will alsovary in stringency. Wash stringency conditions can be defined by saltconcentration and by temperature. As above, wash stringency can beincreased by decreasing salt concentration or by increasing temperature.For example, stringent salt concentration for the wash steps willpreferably be less than about 30 mM NaCl and 3 mM trisodium citrate, andmost preferably less than about 15 mM NaCl and 1.5 mM trisodium citrate.Stringent temperature conditions for the wash steps will ordinarilyinclude a temperature of at least about 25° C., more preferably of atleast about 42° C., and even more preferably of at least about 68° C. Ina preferred embodiment, wash steps will occur at 25° C. in 30 mM NaCl, 3mM trisodium citrate, and 0.1% SDS. In a more preferred embodiment, washsteps will occur at 42 C in 15 mM NaCl, 1.5 mM trisodium citrate, and0.1% SDS. In a more preferred embodiment, wash steps will occur at 68°C. in 15 mM NaCl, 1.5 mM trisodium citrate, and 0.1% SDS. Additionalvariations on these conditions will be readily apparent to those skilledin the art. Hybridization techniques are well known to those skilled inthe art and are described, for example, in Benton and Davis (Science196:180, 1977); Grunstein and Hogness (Proc. Natl. Acad. Sci., USA72:3961, 1975); Ausubel et al. (Current Protocols in Molecular Biology,Wiley Interscience, New York, 2001); Berger and Kimmel (Guide toMolecular Cloning Techniques, 1987, Academic Press, New York); andSambrook et al., Molecular Cloning: A Laboratory Manual, Cold SpringHarbor Laboratory Press, New York.

By “substantially identical” is meant a polypeptide or nucleic acidmolecule exhibiting at least 50% identity to a reference amino acidsequence (for example, any one of the amino acid sequences describedherein) or nucleic acid sequence (for example, any one of the nucleicacid sequences described herein). Preferably, such a sequence is atleast 60%, more preferably 80% or 85%, and more preferably 90%, 95% oreven 99% identical at the amino acid level or nucleic acid to thesequence used for comparison.

Sequence identity is typically measured using sequence analysis software(for example, Sequence Analysis Software Package of the GeneticsComputer Group, University of Wisconsin Biotechnology Center, 1710University Avenue, Madison, Wis. 53705, BLAST, BESTFIT, GAP, orPILEUP/PRETTYBOX programs). Such software matches identical or similarsequences by assigning degrees of homology to various substitutions,deletions, and/or other modifications. Conservative substitutionstypically include substitutions within the following groups: glycine,alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid,asparagine, glutamine; serine, threonine; lysine, arginine; andphenylalanine, tyrosine. In an exemplary approach to determining thedegree of identity, a BLAST program may be used, with a probabilityscore between e⁻³ and e⁻¹⁰⁰ indicating a closely related sequence.

By “subject” is meant a mammal, including, but not limited to, a humanor non-human mammal, such as a bovine, equine, canine, ovine, or feline.

Ranges provided herein are understood to be shorthand for all of thevalues within the range. For example, a range of 1 to 50 is understoodto include any number, combination of numbers, or sub-range from thegroup consisting 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34,35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50.

As used herein, the terms “treat,” treating,” “treatment,” and the likerefer to reducing or ameliorating a disorder and/or symptoms associatedtherewith. It will be appreciated that, although not precluded, treatinga disorder or condition does not require that the disorder, condition orsymptoms associated therewith be completely eliminated.

Unless specifically stated or obvious from context, as used herein, theterm “or” is understood to be inclusive. Unless specifically stated orobvious from context, as used herein, the terms “a”, “an”, and “the” areunderstood to be singular or plural.

Unless specifically stated or obvious from context, as used herein, theterm “about” is understood as within a range of normal tolerance in theart, for example within 2 standard deviations of the mean. About can beunderstood as within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%,0.1%, 0.05%, or 0.01% of the stated value. Unless otherwise clear fromcontext, all numerical values provided herein are modified by the termabout.

The recitation of a listing of chemical groups in any definition of avariable herein includes definitions of that variable as any singlegroup or combination of listed groups. The recitation of an embodimentfor a variable or aspect herein includes that embodiment as any singleembodiment or in combination with any other embodiments or portionsthereof.

Any compositions or methods provided herein can be combined with one ormore of any of the other compositions and methods provided herein.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1D show identification and characterization of6-(4-(diethylamino)-3-nitrophenyl)-5-methyl-4,5-dihydropyridazin-3(2H)-one(DNMDP), a potent and selective cancer cell cytotoxic agent. FIG. 1A isa scatterplot of 1924 compounds showing mean survival of TP53 mutantNCI-H1734 cells, which is a non-small cell lung cancer cell line, andTP53 wild-type A549 cells, another lung cancer cell line, after 48 hoursof treatment at concentrations of 10 μM. DNMDP is indicated with a largearrowhead. Other compounds that selectively killed NCI-H1734 cells areindicated with a small arrow. Positive control staurosporine isindicated with a long arrow. FIG. 1B is a linear graph showing a panelof cell lines that was treated with the indicated concentrations ofDNMDP for 48 hours. FIG. 1C is a linear graph showing the HeLa cell linethat was treated with indicated concentrations of the separatedenantiomers of DNMDP for 48 hours. The (R)-enantiomer had a 500-foldlower EC₅₀ compared to the (S)-enantiomer. FIG. 1D is a structure of(R)-DNMDP.

FIG. 2 shows that DNMDP selectively killed NCI-H1734 and did not affectcell viability in A549. NCI-H1734 and A549 cell lines were treated withindicated compounds and concentrations for 48 hours.

FIG. 3 shows the synthesis scheme of(R)-6-(4-(diethylamino)-3-nitrophenyl)-5-methyl-4,5-dihydropyridazin-3(2H)-one(R)-DNMDP) and analogues. Reaction conditions are as follows: (a) Ac₂O,(91%); (b) 90% HNO₃, H₂SO₄, (19%); (c) NaOH, MeOH/H₂O, (100%), thenCH₃CHO, NaBH(OAc)₃, (7%); (d) (BrCH₂CH₂)₂O, K₂CO₃, DMF, (46%); (e)CH₃CHO, NaBH₃CN, MeOH, (82%).

FIGS. 4A-4C show super-critical fluid (SCF) chromatographs of6-(4-(diethylamino)-3-nitrophenyl)-5-methyl-4,5-dihydropyridazin-3(2H)-one(DNMDP) (top to bottom: ES+, diode array, ES-traces). FIG. 4A are threechromatographs showing Peak 1 (CRO separation); FIG. 4B are threechromatographs showing Peak 2 (CRO separation); FIG. 4C are threechromatographs showing synthesized (R)-DNMDP (5:95 ratio peaks 1:2 byuv).

FIGS. 5A-5C show that Phosphodiesterase 3A (PDE3A) expression correlatedwith sensitivity to6-(4-(diethylamino)-3-nitrophenyl)-5-methyl-4,5-dihydropyridazin-3(2H)-one(DNMDP), but inhibition of PDE3A mediated cAMP hydrolysis did notcorrelate with cytotoxicity. FIG. 5A is a scatterplot showingcorrelation between DNMDP sensitivity and expression of 18,988 genes in766 genomically characterized cell lines. Cell lines were treated for 72hours with concentrations ranging from 66.4 μM-2 nM in 2-fold stepdilutions. The Z-score for Pearson correlation between PDE3A expressionand sensitivity to DNMDP is 8.5. FIG. 5B is a scatterplot showingresults from cell lines from panel A that were treated with 480compounds. DNMDP showed the best correlation between PDE3A expressionand sensitivity. FIG. 5C is a scatterplot showing published PDE3inhibitor IC₅₀ values and EC₅₀ values of HeLa cells treated withindicated compounds up to 10 μM for 48 hours. DNMDP IC₅₀ concentrationfor PDE3A inhibition was determined in FIG. 7B.

FIGS. 6A-6C show chemical structures of6-(4-(diethylamino)-3-nitrophenyl)-5-methyl-4,5-dihydropyridazin-3(2H)-one(DNMDP), siguazodan and levosimendan, respectively.

FIGS. 7A and 7B are graphs showing determination of Phosphodiesterase 3A(PDE3A) in vitro IC₅₀ of6-(4-(diethylamino)-3-nitrophenyl)-5-methyl-4,5-dihydropyridazin-3(2H)-one(DNMDP). FIG. 7A shows PDE3A in vitro inhibition with indicatedconcentrations of positive control trequinsin (IC₅₀ curve was performedby Caliper). FIG. 7B shows PDE3A in vitro inhibition with indicatedconcentrations of DNMDP (IC₅₀ curve was performed by Caliper).

FIGS. 8A and 8B are graphs showing that induction of cAMP signaling didnot phenocopy cytotoxicity induced by6-(4-(diethylamino)-3-nitrophenyl)-5-methyl-4,5-dihydropyridazin-3(2H)-one(DNMDP). Forskolin: FSK. FIG. 8A shows cAMP concentrations that weremeasured 1 hour after treatment with indicated compounds andconcentration in HeLa cells. FIG. 8B shows viability of HeLa cells thatwere treated with indicated compounds and concentrations for 48 hours.

FIGS. 9A-9C show that non-lethal Phosphodiesterase 3 (PDE3) inhibitorsrescued cell death induced by6-(4-(diethylamino)-3-nitrophenyl)-5-methyl-4,5-dihydropyridazin-3(2H)-one(DNMDP) by competing for the binding of PDE3A. FIG. 9A is a scatterplotshowing viability of HeLa cells that were treated with 1600 bioactivecompounds at a concentration of 20 μM in combination with 30 nM (EC70)of DNMDP for 48 hours. The viability was calculated as a percentage ofthe untreated DMSO control. FIG. 9B is a linear graph showing viabilityof HeLa cells that were treated with DNMDP in combination with indicatedconcentrations of non-lethal PDE3 and pan-PDE inhibitors for 48 hours.FIG. 9C shows a SDS-PAGE gel depicting the result of affinitypurification performed on 200 μg of HeLa cell lysate using a DNMDPlinker-analogue tethered to a solid phase with the same rescuecharacteristic as non-lethal PDE3 inhibitors. Indicated compounds wereco-incubated with the linker-analogue. The affinity purified fractionwas run on an SDS-PAGE gel and probed for PDE3A.

FIGS. 10A and 10B show the structure and rescue phenotype oflinker-compound tert-butyl(R)-(2-(2-(2-(ethyl(4-(4-methyl-6-oxo-1,4,5,6-tetrahydropyridazin-3-yl)phenyl)amino)ethoxy)ethoxy)ethyl)carbamate (DNMDP)-2L. FIG. 10A shows the structure ofDNMDP-2L. FIG. 10B is a graph showing the viability of HeLa cells thatwere treated with indicated compounds and concentrations for 48 hours.

FIGS. 11A-11C show that Phosphodiesterase 3A (PDE3A) was not essentialin sensitive cell lines, but was required for relaying the cytotoxicsignal. FIG. 11A is a Western blot. HeLa cells were infected with Cas9and indicated guide RNAs (sgRNA) against PDE3A. Western blots wereprobed for PDE3A at indicated time points. FIG. 11B is a bar graphshowing percent rescue of HeLa cells that were infected with indicatedsgRNAs for two weeks and treated with 1 μM of6-(4-(diethylamino)-3-nitrophenyl)-5-methyl-4,5-dihydropyridazin-3(2H)-one(DNMDP) for 48 hours. Percent rescue was normalized to the Cas9-onlycontrol. FIG. 11C is a plot showing viability of cells infected withindicated sgRNAs and treated with various concentrations of6-(4-(diethylamino)-3-nitrophenyl)-5-methyl-4,5-dihydropyridazin-3(2H)-one(DNMDP).

FIGS. 12A and 12B are a Western blot and a graph showing that reductionof Phosphodiesterase 3A (PDE3A) protein level caused resistance to6-(4-(diethylamino)-3-nitrophenyl)-5-methyl-4,5-dihydropyridazin-3(2H)-one(DNMDP). In FIG. 12A HeLa cells were treated with scrambled controlsiRNA or a combination of four different siRNAs targeting PDE3A. Cellswere lysed at indicated time-points and immunoblotted for PDE3A andActin. FIG. 12B is a linear graph showing viability of HeLa cells thatwere treated with indicated concentrations of DNMDP analogue 3 for 48hours.

FIGS. 13A-13C show that Phosphodiesterase 3A (PDE3A) immunoprecipitationin the presence of6-(4-(diethylamino)-3-nitrophenyl)-5-methyl-4,5-dihydropyridazin-3(2H)-one(DNMDP) revealed novel SIRT7 and SLFN12 interaction. FIG. 13A shows aschematic overview of the affinity enrichment followed by quantitativeproteomics of PDE3A performed in HeLa cells. All cells were treated forfour hours prior to lysis with 10 μM of indicated compounds. Thepresence of all compounds was maintained throughout the experimentincluding washing steps. FIG. 13B is a scatterplot showing log₂ ratiosfor proteins that were enriched in anti-PDE3A immuno precipitates in theDMSO treated HeLa cells compared to anti-PDE3A immuno precipitates inthe presence of blocking peptide specific to the PDE3A antibody; eachdot represents a protein. FIG. 13C is a scatterplot showing Log₂ ratiosof changes of proteins bound to PDE3A in the presence of DNMDP versustrequinsin. Each dot represents the average of two replicates percondition for an individual protein. In all cases, the data plottedpassed the Bland-Altman test with 95% confidence interval forreproducibility.

FIGS. 14A-14C show results of replicate PDE3A-protein interactionstudies using PDE3A as bait under different conditions. Each scatterplotshowed log₂ ratios of two replicates for proteins that were enriched byPDE3A under different conditions over enrichment by PDE3A in thepresence of blocking peptide. Each dot represents the log₂ ratio forthat particular protein, medium gray dots correspond to aBenjamini-Hochberg adjusted p value <0.01, light gray dots representproteins that fall outside of the Blandt-Altman test for reproducibilitywithin a 95% confidence interval. In FIG. 14A protein enrichment wasaccomplished by immunoprecipitation using anti-PDE3A. In FIG. 14Bprotein enrichment was accomplished by immunoprecipitation usinganti-PDE3A in the presence of DNMDP. In FIG. 14C protein enrichment wasaccomplished by immunoprecipitation using anti-PDE3A in the presence oftrequinsin.

FIGS. 15A-15E show that cell lines with dual expression of SLFN12 andPDE3A were significantly enriched for DNMDP-sensitive cell lines. FIG.15A is a scatterplot showing mRNA robust multichip average (RMA)expression values for PDE3A and SLFN12 from the Cancer Cell LineEncyclopedia (CCLE) database (a detailed genetic characterization of alarge panel of human cancer cell lines) with sensitive cell linesindicated (Barretina et al., Nature 483, 603-607, 2012). 21 sensitivecell lines were binned in three groups of 7 based on area under thecurve (AUC) rank. FIG. 15B is a bar graph showing results of a Fisher'sexact test on DNMDP sensitivity of cell lines with high expression ofboth SLFN12 and PDE3A (RMA Log 2>5) compared to other cell lines. Thetop half of the bar on the right indicates melanoma cell lines. FIG. 15Cis a scatterplot showing mRNA RPKM+1 expression values for PDE3A andSLFN12 from RNA sequencing data. FIG. 15D is a bar graph showing qPCRexpression changes of SLFN12 in HeLa cells transduced with shSLFN12normalized to GAPDH. FIG. 15E is a plot showing viability of HeLa cellstransduced with indicated shRNA reagents and treated with indicatedconcentrations of DNMDP for 72 hours.

FIGS. 16A and 16B are scatter plots showing that SLFN12 expression wasamongst the top genes correlating with DNMDP sensitivity. FIG. 16A showsthe correlation between DNMDP sensitivity and expression of 18,988 genesin 766 genomically characterized cell lines. Cell lines were treated for72 hours with concentrations ranging from 66.4 μM-2 nM in 2-fold stepdilutions. FIG. 16B is a scatterplot showing a correlation between DNMDPsensitivity and expression of 18,988 genes in 766 genomicallycharacterized cell lines. Expression levels were corrected for PDE3Aexpression as described earlier (Kim et al., Genetica 131, 151-156,2007). Cell lines were treated for 72 hours with concentrations rangingfrom 66.4 μM-2 nM in 2-fold step dilutions.

FIGS. 17A-7B show that DNMDP induces apoptosis in HeLa cells. FIG. 17Ais a plot showing viability of HeLa cells treated for 48 hours withindicated concentrations of DNMDP. Caspase-Glo represents Caspase 3/7activity indicating induction of apoptosis. CellTiter-Glo reflectsviability. FIG. 17B is an immunoblot. HeLa cells were treated for 36hours with indicated compounds and concentrations. HeLa cells wereharvested and immunoblotted for PARP-cleavage products, indicative ofapoptosis.

FIG. 18 is a scatterplot of PDE3A mRNA expression and sensitivity toDNMDP of 766 cancer cell lines.

FIG. 19 is an immunoblot showing that DNMDP induces interaction betweenPDE3A and SIRT7 and SLFN12 in HeLa cells. HeLa cells were transfectedwith indicated plasmids and treated with indicated compounds with afinal concentration of 10 μM for four hours. Endogenous PDE3A wasimmunoprecipitated and immunoblotted for V5 to identify novelinteraction with SIRT7 and SLFN12 (upper two panels). Immunoprecipitateinput was immunoblotted for PDE3A and V5 (lower two panels). V5-SLFN12was undetectable in whole cell lysate.

FIG. 20 is an immunoblot showing confirmation of mass spectrometricresults herein using affinity reagents. FIG. 20 shows that DNMDP and(weakly) anagrelide, but not trequinsin, induced PDE3A and SFLN12complex formation.

FIG. 21 is a set of tables showing that SLFN12 is lost in cells thathave acquired resistance to DNMDP.

FIG. 22 is a plot showing sensitization of a DNMDP-resistant cell lineby expression of SLFN12 or expression of SFLN12 and PDE3A.

FIG. 23 is a scatter plot showing sensitivity of Leiomyosarcomas (LMS)to PDE3A modulation based on SLFN12 expression level.

Table 1 shows sensitivity data of 766 cancer cell lines treated withDNMDP. Cell lines were treated for 72 hours with concentrations rangingfrom 66.4 μM-2 nM in 2-fold step dilutions.

Table 2 shows results from panel of 19 phosphodiesterase inhibitionreactions performed by Caliper. DNMDP concentration was 100 nM.

Table 3 shows RPKM values of SLFN12 and PDE3A expression in multiplehealthy tissue types.

Table 4 showing Leiomyosarcoma sensitivity to DNMDP

Table 5 shows binding of DNMDP to PDE3A(677-1141).

Compositions and articles defined by the invention were isolated orotherwise manufactured in connection with the examples provided below.Other features and advantages of the invention will be apparent from thedetailed description, and from the claims.

DETAILED DESCRIPTION

As described below, the present invention features improved methods ofidentifying patients having cancer (e.g., melanoma, endometrium, lung,hematopoetic/lymphoid, ovarian, cervical, soft-tissue sarcoma,leiomyosarcoma, urinary tract, pancreas, thyroid, kidney, glioblastoma,or breast cancer)) that is sensitive to treatment with aphosphodiesterase 3A (PDE3A) modulator by detecting co-expression ofPDE3A and Schlafen 12 (SLFN12) polypeptides or polynucleotides in acancer cell derived from such patients. The invention is based at leastin part on the discovery that sensitivity to phosphodiesterase 3Amodulators, such as6-(4-(diethylamino)-3-nitrophenyl)-5-methyl-4,5-dihydropyridazin-3(2H)-one,or DNMDP, in 766 cancer cell lines correlated with expression of thephosphodiesterase 3A gene, PDE3A. Like DNMDP, a subset of PDE3Ainhibitors kill selected cancer cells while others do not; thesecell-sparing PDE3A inhibitors instead block DNMDP induced cytotoxicity.Furthermore, PDE3A depletion leads to DNMDP resistance. DNMDP binding toPDE3A promotes an interaction between PDE3A and Sirtuin 7 (SIRT7) andSchlafen 12 (SLFN12), suggesting a neomorphic activity, and SLFN12 andPDE3A co-expression correlated with DNMDP sensitivity. These resultsindicate that PDE3A modulators are promising cancer therapeutic agentsand demonstrate the power of chemogenomics in small-molecule discoveryand target-identification.

Accordingly, the invention provides methods of selecting a subject ashaving a cancer that responds to a PDE3A modulator, where the selectionmethod involves detecting co-expression of PDE3A and Schlafen 12(SLFN12) polypeptides or polynucleotides, in a cancer cell derived fromsuch subjects.

PDE3A Modulator

The identification of PDE3A modulators was made in connection with aphenotypic screen designed to identify cytotoxic small molecules in amutant tp53 background. A chemogenomics approach complementstarget-driven drug development programs, which consists of extensive invitro and in vivo target validation, and can also be referred to asreverse chemogenomics (Zheng et al., Curr Issues Mol Biol 4, 33-43,2002). Many U.S. Food and Drug Administration (FDA)-approved targetedtherapies have been developed this way, among them small-molecule kinaseinhibitors that target oncogenic somatic driver mutations (Moffat etal., Nat Rev Drug Discov 13, 588-602, 2014). However, the discovery anddevelopment of targeted therapies is often hampered by limitations inknowledge of the biological function of the target, its mechanism ofaction, and the available chemical matter to selectively inhibit thetarget.

Phenotypic screening can discover novel targets for cancer therapy whosespecific molecular mechanism is often elucidated by future studies(Swinney et al., Nat Rev Drug Discov 10, 507-519, 2011). In recentyears, two classes of anti-cancer drugs found by unbiased phenotypicscreening efforts have been approved by the FDA. Lenalidomide andpomalidomide were found to be modulators of an E3-ligase that alter theaffinity of its target, leading to degradation of lineage specifictranscription factors (Krönke et al., Science 343, 301-305, 2014; Lu etal., Science 343, 305-309, 2014), whereas romidepsin and vorinostat werelater identified as histone deacetylase (HDAC) inhibitors (Moffat etal., Nat Rev Drug Discov 13, 588-602, 2014; Nakajima et al., Exp. CellRes. 241, 126-133, 1998, Marks et al., Nat Biotechnol 25, 84-90, 2007).

Tumor suppressor alterations are suitable targets for phenotypicscreening as they are not directly targetable with small molecules,although synthetic lethal approaches such as olaparib treatment ofBRCA1/BRCA2 mutant cancers have proven to be effective. According tocurrent knowledge, the tp53 tumor suppressor gene is the most frequentlymutated across human cancer, with somatic mutations detected in 36% of4742 cancers subjected to whole exome sequencing. Despite many attempts,no compounds that selectively kill tp53 mutant cells have beenidentified.

A phenotypic screen developed to identify small molecules causingsynthetic lethality in tp53 mutant cancer cells enabled theserendipitous discovery of a class of cancer-selective cytotoxic agentswhich act as modulators of phosphodiesterase 3A (PDE3A), as describedherein below. Cyclic nucleotide phosphodiesterases catalyze thehydrolysis of second messenger molecules cyclic adenosine monophosphate(cAMP) and cyclic guanosine monophosphate (cGMP), and are important inmany physiological processes. Several phosphodiesterase inhibitors havebeen approved for clinical treatment, including PDE3 inhibitorsmilrinone, cilostazol, and levosimendan for cardiovascular indicationsand inhibition of platelet coagulation, as well as the PDE3 inhibitoranagrelide for thrombocythemia. PDE5 inhibitors, e.g. vardenafil, areused for smooth muscle disorders including erectile dysfunction andpulmonary arterial hypertension, and the PDE4 inhibitor roflumilastreduces exacerbations from chronic obstructive pulmonary disease (COPD).

Phosphodiesterase inhibitors act by direct inhibition of their targetsor by allosteric modulation; for example, structural analysis of PDE4has led to the design of PDE4D and PDE4B allosteric modulators (Burginet al., Nat Biotechnol 28, 63-70, 2010; Gurney et al., Neurotherapeutics12, 49-56, 2015). The data provided herein below indicates that thecancer cytotoxic phosphodiesterase modulator DNMDP likely acts through asimilar allosteric mechanism.

Accordingly, the invention provides methods for identifying subjectsthat have a malignancy that is likely to respond to PDE3A modulatortreatment based on the level of PDE3A and SLFN12 expression in a subjectbiological sample comprising a cancer cell. In some embodiments, thePDE3A modulator is DNMDP. In some other embodiments, the PDE3A modulatoris anagrelide or zardaverine.

Compound Forms and Salts

The compounds of the present invention include the compounds themselves,as well as their salts and their prodrugs, if applicable. A salt, forexample, can be formed between an anion and a positively chargedsubstituent (e.g., amino) on a compound described herein. Suitableanions include chloride, bromide, iodide, sulfate, nitrate, phosphate,citrate, methanesulfonate, trifluoroacetate, and acetate. Likewise, asalt can also be formed between a cation and a negatively chargedsubstituent (e.g., carboxylate) on a compound described herein. Suitablecations include sodium ion, potassium ion, magnesium ion, calcium ion,and an ammonium cation such as tetramethylammonium ion. Examples ofprodrugs include C₁₋₆ alkyl esters of carboxylic acid groups, which,upon administration to a subject, are capable of providing activecompounds.

Pharmaceutically acceptable salts of the compounds of the presentdisclosure include those derived from pharmaceutically acceptableinorganic and organic acids and bases. As used herein, the term“pharmaceutically acceptable salt” refers to a salt formed by theaddition of a pharmaceutically acceptable acid or base to a compounddisclosed herein. As used herein, the phrase “pharmaceuticallyacceptable” refers to a substance that is acceptable for use inpharmaceutical applications from a toxicological perspective and doesnot adversely interact with the active ingredient.

Examples of suitable acid salts include acetate, adipate, alginate,aspartate, benzoate, benzenesulfonate, bisulfate, butyrate, citrate,camphorate, camphorsulfonate, digluconate, dodecylsulfate,ethanesulfonate, formate, fumarate, glucoheptanoate, glycolate,hemisulfate, heptanoate, hexanoate, hydrochloride, hydrobromide,hydroiodide, 2-hydroxyethanesulfonate, lactate, maleate, malonate,methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, palmoate,pectinate, persulfate, 3-phenylpropionate, phosphate, picrate, pivalate,propionate, salicylate, succinate, sulfate, tartrate, thiocyanate,tosylate and undecanoate. Other acids, such as oxalic, while not inthemselves pharmaceutically acceptable, may be employed in thepreparation of salts useful as intermediates in obtaining the compoundsof the present invention and their pharmaceutically acceptable acidaddition salts. Salts derived from appropriate bases include alkalimetal (e.g., sodium), alkaline earth metal (e.g., magnesium), ammoniumand N-(alkyl)₄ ⁺ salts. The present invention also envisions thequaternization of any basic nitrogen-containing groups of the compoundsdisclosed herein. Water or oil-soluble or dispersible products may beobtained by such quaternization. Salt forms of the compounds of any ofthe formulae herein can be amino acid salts of carboxyl groups (e.g.,L-arginine, -lysine, -histidine salts).

Lists of suitable salts are found in Remington's PharmaceuticalSciences, 17th ed., Mack Publishing Company, Easton, Pa., 1985, p. 1418;Journal of Pharmaceutical Science, 66, 2 (1977); and “PharmaceuticalSalts: Properties, Selection, and Use A Handbook; Wermuth, C. G. andStahl, P. H. (eds.) Verlag Helvetica Chimica Acta, Zurich, 2002 [ISBN3-906390-26-8] each of which is incorporated herein by reference intheir entireties.

The neutral forms of the compounds may be regenerated by contacting thesalt with a base or acid and isolating the parent compound in theconventional manner. The parent form of the compound differs from thevarious salt forms in certain physical properties, such as solubility inpolar solvents, but otherwise the salts are equivalent to the parentform of the compound for the purposes of the present invention.

In addition to salt forms, the present invention provides compoundswhich are in a prodrug form. Prodrugs of the compounds described hereinare those compounds that undergo chemical changes under physiologicalconditions to provide the compounds of the present invention.Additionally, prodrugs can be converted to the compounds of the presentinvention by chemical or biochemical methods in an ex vivo environment.For example, prodrugs can be slowly converted to the compounds of thepresent invention when placed in a transdermal patch reservoir with asuitable enzyme or chemical reagent. Prodrugs are often useful because,in some situations, they may be easier to administer than the parentdrug. They may, for instance, be more bioavailable by oraladministration than the parent drug. The prodrug may also have improvedsolubility in pharmacological compositions over the parent drug. A widevariety of prodrug derivatives are known in the art, such as those thatrely on hydrolytic cleavage or oxidative activation of the prodrug. Anexample, without limitation, of a prodrug would be a compound of thepresent invention which is administered as an ester (the “prodrug”), butthen is metabolically hydrolyzed to the carboxylic acid, the activeentity. Additional examples include peptidyl derivatives of a compoundof the present invention.

The present invention also includes various hydrate and solvate forms ofthe compounds.

The compounds of the present invention may also contain unnaturalproportions of atomic isotopes at one or more of the atoms thatconstitute such compounds. For example, the compounds may beradiolabeled with radioactive isotopes, such as for example tritium(³H), iodine-125 (¹²⁵I) or carbon-14 (¹⁴C). All isotopic variations ofthe compounds of the present invention, whether radioactive or not, areintended to be encompassed within the scope of the present invention.

Diagnostics

The present invention features diagnostic assays for thecharacterization of cancer. In one embodiment, levels of PDE3A and/orSchlafen 12 (SLFN12) polynucleotides or polypeptides are measured in asubject sample and used as an indicator of cancer that is responsive totreatment with a PDE3A modulator. Levels of PDE3A and/or Schlafen 12polynucleotides may be measured by standard methods, such asquantitative PCR, Northern Blot, microarray, mass spectrometry, and insitu hybridization. Standard methods may be used to measure levels ofPDE3A and/or Schlafen 12, polypeptides in a biological sample derivedfrom a tumor. Such methods include immunoassay, ELISA, western blottingusing an antibody that binds PDE3A and/or Schlafen 12 andradioimmunoassay. Elevated levels of PDE3A and Schlafen 12polynucleotides or polypeptides relative to a reference are considered apositive indicator of cancer that is responsive to treatment with aPDE3A modulator.

Types of Biological Samples

In characterizing the responsiveness of a malignancy in a subject toPDE3A modulator treatment, the level of PDE3A and/or SLFN12 expressionis measured in different types of biologic samples. In one embodiment,the biologic sample is a tumor sample.

PDE3A and/or SLFN12 expression is higher in a sample obtained from asubject that is responsive to PDE3A modulator treatment than the levelof expression in a non-responsive subject. In another embodiment, PDE3Aand/or SLFN12 is at least about 5, 10, 20, or 30-fold higher in asubject with a malignancy than in a healthy control. Fold change valuesare determined using any method known in the art. In one embodiment,change is determined by calculating the difference in expression ofPDE3A and/or SLFN12 in a cancer cell vs the level present in anon-responsive cancer cell or the level present in a correspondinghealthy control cell.

Selection of a Treatment Method

As reported herein below, subjects suffering from a malignancy may betested for PDE3A and/or SLFN12 expression in the course of selecting atreatment method. Patients characterized as having increased PDE3Aand/or SLFN12 relative to a reference level are identified as responsiveto PDE3A modulator treatment.

Kits

The invention provides kits for characterizing the responsiveness orresistance of a subject to PDE3A modulator treatment.

Also provided herein are kits that can include a therapeutic compositioncontaining an effective amount of a PDE3A modulator in, e.g., unitdosage form.

In one embodiment, a diagnostic kit of the invention provides a reagentfor measuring relative expression of PDE3A and SLFN12. Such reagentsinclude capture molecules (e.g., antibodies that recognize PDE3A andSLFN12 polypeptides or nucleic acid probes that hybridize with PDE3A andSLFN12 polynucleotides).

In some embodiments, the kit comprises a sterile container whichincludes a therapeutic or diagnostic composition; such containers can beboxes, ampoules, bottles, vials, tubes, bags, pouches, blister-packs, orother suitable container forms known in the art. Such containers can bemade of plastic, glass, laminated paper, metal foil, or other materialssuitable for holding medicaments.

In one embodiment, a kit of the invention comprises reagents formeasuring PDE3A and/or SLFN12 levels. If desired, the kit furthercomprises instructions for measuring PDE3A and/or SLFN12 and/orinstructions for administering the PDE3A modulator to a subject having amalignancy, e.g., a malignancy selected as responsive to PDE3A modulatortreatment. In particular embodiments, the instructions include at leastone of the following: description of the therapeutic agent; dosageschedule and administration for treatment or prevention of malignancy orsymptoms thereof; precautions; warnings; indications;counter-indications; over dosage information; adverse reactions; animalpharmacology; clinical studies; and/or references. The instructions maybe printed directly on the container (when present), or as a labelapplied to the container, or as a separate sheet, pamphlet, card, orfolder supplied in or with the container.

The practice of the present invention employs, unless otherwiseindicated, conventional techniques of molecular biology (includingrecombinant techniques), microbiology, cell biology, biochemistry andimmunology, which are well within the purview of the skilled artisan.Such techniques are explained fully in the literature, such as,“Molecular Cloning: A Laboratory Manual”, second edition (Sambrook,1989); “Oligonucleotide Synthesis” (Gait, 1984); “Animal Cell Culture”(Freshney, 1987); “Methods in Enzymology” “Handbook of ExperimentalImmunology” (Weir, 1996); “Gene Transfer Vectors for Mammalian Cells”(Miller and Calos, 1987); “Current Protocols in Molecular Biology”(Ausubel, 1987); “PCR: The Polymerase Chain Reaction”, (Mullis, 1994);“Current Protocols in Immunology” (Coligan, 1991). These techniques areapplicable to the production of the polynucleotides and polypeptides ofthe invention, and, as such, may be considered in making and practicingthe invention. Particularly useful techniques for particular embodimentswill be discussed in the sections that follow.

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how tomake and use the assay, screening, and therapeutic methods of theinvention, and are not intended to limit the scope of the invention.

EXAMPLES Example 1. Identification of a Cell-Selective Cytotoxic SmallMolecule

To identify anti-cancer compounds with cell-selective cytotoxicactivity, an unbiased chemical screen was performed in two lungadenocarcinoma cell lines, A549 and NCI-H1734, both of which harboroncogenic KRAS mutations and truncating STK11 mutations, and which wereTP53 wild type and mutant (R273L), respectively. 1,924 compounds werescreened from the Molecular Libraries Small-Molecule Repositoryvalidation set in the A549 and NCI-H1734 cell lines at a singleconcentration of 10 μM in 384-well format in duplicate. As a proxy forcellular viability, ATP content was measured after 48 hours of compoundtreatment.

Three compounds showed a selective reduction in cell viability for theNCI-H1734 cell line compared to the A549 cell line, with anapproximately 50% reduction in the NCI-H1734 cell line, which is >4median absolute deviations from the median in the negative direction,compared to a minimal change of <1 median absolute deviations from themedian in the A549 cell line (FIG. 1A). Retesting the three compounds ina dose-response analysis validated that one compound,6-(4-(diethylamino)-3-nitrophenyl)-5-methyl-4,5-dihydropyridazin-3(2H)-one,or DNMDP, was specifically toxic to the NCI-H1734 cell line (FIG. 2).

Testing of additional cell lines with DNMDP showed clear cell-selectivecytotoxicity, with an EC₅₀ between 10 and 100 nM for two additional lungadenocarcinoma cell lines, NCI-H1563 and NCI-H2122, and for HeLacervical carcinoma cells, but an EC₅₀ greater than 1 μM for A549, MCF7,and PC3 cells (FIG. 1B; FIG. 1C). Caspase activity was detected by acaspase-sensitive luciferase assay and by poly ADP ribose polymerase(PARP) cleavage in HeLa cells upon DNMDP treatment, indicating thatsensitive cells undergo apoptosis after DNMDP exposure (FIGS. 17A-17B).To characterize cellular sensitivity to DNMDP further, 766 genomicallycharacterized cancer cell lines were screened for DMNDP sensitivity atconcentrations ranging from 66.4 μM to 2 nM in 2-fold dilution steps for72 hours. From these cell lines, 22 cell lines were categorized assensitive with a robust Z-score lower than −4, which representedmultiple lineages including multiple melanoma cell lines, amongst others(Table 1).

Next, the DNMDP enantiomers were separated by chiral super-criticalfluid (SCF) chromatography. One enantiomer was 500-fold more potent inHeLa cells than the other (FIGS. 1C and D). The (R)-enantiomer wassynthesized from commercially available starting materials (FIG. 3).This synthesized enantiomer had similar activity to the more potentseparated material and was identical by chiral SCF chromatography,confirming stereochemistry of the active enantiomer (FIGS. 4A-4C). Two(R)-des-nitro analogues of DNMDP were synthesized, both of which testedsimilarly to (R)-DNMDP (FIG. 3). FIGS. 4A-4C show super-critical fluid(SCF) chromatographs of6-(4-(diethylamino)-3-nitrophenyl)-5-methyl-4,5-dihydropyridazin-3(2H)-one(DNMDP) (top to bottom: ES+, diode array, ES-traces). FIG. 4A shows Peak1 (CRO separation); FIG. 4B shows Peak 2 (CRO separation); and FIG. 4Cshows synthesized (R)-DNMDP (5:95 ratio peaks 1:2 by uv).

TABLE 1 Sensitivity data of 766 cancer cell lines treated with DNMDPDNMDP Cell line Lineage AUC Robust Z-score COV318 OVARY 0.095838−6.863450362 IGR37 SKIN 0.41146 −6.532158389 JHUEM1 ENDOMETRIUM 0.53468−6.402820773 HEL HAEMATOPOIETIC AND 0.57955 −6.355723071 LYMPHOID TISSUECORL51 LUNG 0.59436 −6.340177786 HEL9217 HAEMATOPOIETIC AND 0.75005−6.176758102 LYMPHOID TISSUE NCIH1563 LUNG 1.0887 −5.821294837 SKMEL3SKIN 1.2215 −5.681901594 NCIH2122 LUNG 1.3105 −5.58848293 RVH421 SKIN1.4556 −5.436179018 HUT78 HAEMATOPOIETIC AND 1.5307 −5.35735046 LYMPHOIDTISSUE DKMG CENTRAL NERVOUS SYSTEM 1.7217 −5.156867709 GB1 CENTRALNERVOUS SYSTEM 1.8269 −5.046444748 G292CLONEA141B1 BONE 1.9664−4.900018865 HMCB SKIN 1.9762 −4.889732315 A2058 SKIN 2.0833−4.777315024 NCIH1734 LUNG 2.2179 −4.636032415 NCIH196 LUNG 2.5263−4.312320999 LI7 LIVER 2.5414 −4.296471315 JHOM1 OVARY 2.7006−4.129367368 COLO741 SKIN 2.7231 −4.10575029 HS578T BREAST 2.8012−4.023772788 K029AX SKIN 2.9362 −3.88207032 MONOMAC1 HAEMATOPOIETIC AND2.9692 −3.847431939 LYMPHOID TISSUE HT1197 URINARY TRACT 3.0929−3.717590492 NCIH520 LUNG 3.1351 −3.67329535 CAL78 BONE 3.1711−3.635508025 NCIH647 LUNG 3.2187 −3.585544785 CGTHW1 THYROID 3.4296−3.36417404 NCIH1666 LUNG 3.6097 −3.175132451 L33 PANCREAS 3.625−3.159072838 UACC62 SKIN 3.9116 −2.858243747 CAS1 CENTRAL NERVOUS SYSTEM3.9993 −2.766189625 CAL51 BREAST 4.0017 −2.76367047 OSRC2 KIDNEY 4.326−2.423269652 X8505C THYROID 4.3418 −2.406685215 SH4 SKIN 4.3672−2.380024158 NCIH1395 LUNG 4.4473 −2.29594736 SNU503 LARGE INTESTINE4.5692 −2.16799528 HS729 SOFT TISSUE 4.6518 −2.081294362 SW579 THYROID4.697 −2.033850277 YH13 CENTRAL NERVOUS SYSTEM 4.7007 −2.029966579DBTRG05MG CENTRAL NERVOUS SYSTEM 4.7415 −1.987140944 SEM HAEMATOPOIETICAND 4.7433 −1.985251578 LYMPHOID TISSUE HS852T SKIN 4.7511 −1.977064324SNU449 LIVER 4.752 −1.976119641 NCIH2286 LUNG 4.7782 −1.948618866 JHOS2OVARY 4.8254 −1.899075485 BICR31 UPPER AERODIGESTIVE 4.8356 −1.888369076TRACT IGR1 SKIN 4.8613 −1.861393125 JHUEM3 ENDOMETRIUM 4.93 −1.789282313SNU387 LIVER 4.9639 −1.753699249 UMUC1 URINARY TRACT 4.9933 −1.7228396X8305C THYROID 5.0004 −1.7153871 NCIH1915 LUNG 5.0031 −1.712553051P31FUJ HAEMATOPOIETIC AND 5.0106 −1.704680691 LYMPHOID TISSUE COLO678LARGE INTESTINE 5.0245 −1.690090585 EOL1 HAEMATOPOIETIC AND 5.0478−1.665633789 LYMPHOID TISSUE KNS42 CENTRAL NERVOUS SYSTEM 5.0791−1.632779809 SW1783 CENTRAL NERVOUS SYSTEM 5.1161 −1.593942837 HS940TSKIN 5.1573 −1.550697343 SNU685 ENDOMETRIUM 5.206 −1.499579489 BCPAPTHYROID 5.2336 −1.470609207 COLO829 SKIN 5.2432 −1.460532587 DM3 PLEURA5.2635 −1.439224734 OCUM1 STOMACH 5.2843 −1.417392058 M059K CENTRALNERVOUS SYSTEM 5.3059 −1.394719663 MG63 BONE 5.3943 −1.301930788NCIH2172 LUNG 5.4245 −1.270231421 CAOV3 OVARY 5.4646 −1.228140539 PEERHAEMATOPOIETIC AND 5.4754 −1.216804342 LYMPHOID TISSUE HS839T SKIN5.5232 −1.166631172 CORL105 LUNG 5.5442 −1.144588566 SNU5 STOMACH 5.5498−1.138710537 MFE296 ENDOMETRIUM 5.5618 −1.126114762 NCIH854 LUNG 5.576−1.111209762 NCIH146 LUNG 5.5773 −1.10984522 NCIH2081 LUNG 5.5811−1.105856558 COV644 OVARY 5.5849 −1.101867896 VCAP PROSTATE 5.5863−1.100398388 BICR18 UPPER AERODIGESTIVE 5.6 −1.086018212 TRACT RH18 SOFTTISSUE 5.6283 −1.056313176 KPNYN AUTONOMIC GANGLIA 5.6717 −1.010758457KPNSI9S AUTONOMIC GANGLIA 5.6827 −0.99921233 SKCO1 LARGE INTESTINE 5.688−0.993649196 MV411 HAEMATOPOIETIC AND 5.6905 −0.991025076 LYMPHOIDTISSUE COV362 OVARY 5.6913 −0.990185358 NCO2 HAEMATOPOIETIC AND 5.7088−0.971816519 LYMPHOID TISSUE JHH4 LIVER 5.71 −0.970556942 NCIH2141 LUNG5.7218 −0.958171096 LXF289 LUNG 5.734 −0.945365392 MEWO SKIN 5.738−0.9411668 TE125T SOFT TISSUE 5.744 −0.934868913 SNU869 BILIARY TRACT5.7543 −0.924057539 LNCAPCLONEFGC PROSTATE 5.7557 −0.922588032 NCIH2009LUNG 5.7594 −0.918704335 SKNBE2 AUTONOMIC GANGLIA 5.7717 −0.905793666IALM LUNG 5.775 −0.902329827 DU145 PROSTATE 5.7825 −0.894457468 HCC1419BREAST 5.7835 −0.89340782 NALM6 HAEMATOPOIETIC AND 5.7872 −0.889524123LYMPHOID TISSUE PECAPJ15 UPPER AERODIGESTIVE 5.789 −0.887634757 TRACTLU99 LUNG 5.8016 −0.874409193 LAMA84 HAEMATOPOIETIC AND 5.8201−0.854990707 LYMPHOID TISSUE ONCODG1 OVARY 5.8296 −0.845019051 HS888TBONE 5.8353 −0.839036058 SKNSH AUTONOMIC GANGLIA 5.8424 −0.831583558TUHR14TKB KIDNEY 5.8451 −0.828749509 PF382 HAEMATOPOIETIC AND 5.8519−0.821611903 LYMPHOID TISSUE ALLSIL HAEMATOPOIETIC AND 5.8724−0.800094121 LYMPHOID TISSUE KMS34 HAEMATOPOIETIC AND 5.8799−0.792221762 LYMPHOID TISSUE BICR6 UPPER AERODIGESTIVE 5.8837−0.788233099 TRACT GRANTA519 HAEMATOPOIETIC AND 5.8937 −0.77773662LYMPHOID TISSUE OCIAML2 HAEMATOPOIETIC AND 5.8945 −0.776896902 LYMPHOIDTISSUE SUIT2 PANCREAS 5.8956 −0.775742289 BT549 BREAST 5.9226−0.747401796 KMS28BM HAEMATOPOIETIC AND 5.9369 −0.732391831 LYMPHOIDTISSUE HCC1428 BREAST 5.9402 −0.728927992 HCC1500 BREAST 5.9451−0.723784718 A549 LUNG 5.9509 −0.71769676 KCL22 HAEMATOPOIETIC AND5.9598 −0.708354893 LYMPHOID TISSUE COLO679 SKIN 5.9634 −0.704576161SKMEL5 SKIN 5.9639 −0.704051337 HCC1395 BREAST 5.9716 −0.695969048NCIH1435 LUNG 5.9756 −0.691770456 LOUNH91 LUNG 5.9793 −0.687886759RPMI8402 HAEMATOPOIETIC AND 5.9827 −0.684317956 LYMPHOID TISSUE COLO668LUNG 5.9969 −0.669412956 SKLU1 LUNG 6.0109 −0.654717885 KMS12BMHAEMATOPOIETIC AND 6.0135 −0.6519888 LYMPHOID TISSUE SNU1272 KIDNEY6.0226 −0.642437004 MOLM6 HAEMATOPOIETIC AND 6.0447 −0.619239786LYMPHOID TISSUE EPLC272H LUNG 6.0469 −0.61693056 SCC4 UPPERAERODIGESTIVE 6.0502 −0.613466722 TRACT LMSU STOMACH 6.0528 −0.610737638KMS20 HAEMATOPOIETIC AND 6.0542 −0.60926813 LYMPHOID TISSUE G402 SOFTTISSUE 6.0606 −0.602550384 KYSE410 OESOPHAGUS 6.0741 −0.588380137 L540HAEMATOPOIETIC AND 6.0807 −0.581452461 LYMPHOID TISSUE MOLT13HAEMATOPOIETIC AND 6.084 −0.577988623 LYMPHOID TISSUE L1236HAEMATOPOIETIC AND 6.0853 −0.57662408 LYMPHOID TISSUE LP1 HAEMATOPOIETICAND 6.1029 −0.558150277 LYMPHOID TISSUE SNU620 STOMACH 6.1039−0.557100629 MALME3M SKIN 6.112 −0.548598481 GSU STOMACH 6.1172−0.543140312 MCF7 BREAST 6.1256 −0.53432327 COLO800 SKIN 6.1272−0.532643833 MKN7 STOMACH 6.1453 −0.513645206 SNU119 OVARY 6.1473−0.51154591 U118MG CENTRAL NERVOUS SYSTEM 6.1481 −0.510706192 OCILY19HAEMATOPOIETIC AND 6.1512 −0.507452283 LYMPHOID TISSUE RKN SOFT TISSUE6.1579 −0.500419642 DV90 LUNG 6.1676 −0.490238057 NCIH1355 LUNG 6.171−0.486669254 KMM1 HAEMATOPOIETIC AND 6.1723 −0.485304712 LYMPHOID TISSUENCIH1184 LUNG 6.1776 −0.479741578 U937 HAEMATOPOIETIC AND 6.1777−0.479636613 LYMPHOID TISSUE EJM HAEMATOPOIETIC AND 6.1782 −0.479111789LYMPHOID TISSUE C32 SKIN 6.1786 −0.47869193 NCIH23 LUNG 6.1854−0.471554324 RERFLCAD1 LUNG 6.1862 −0.470714606 T3M10 LUNG 6.1867−0.470189782 U266B1 HAEMATOPOIETIC AND 6.1906 −0.466096155 LYMPHOIDTISSUE CAL54 KIDNEY 6.1949 −0.461582669 DND41 HAEMATOPOIETIC AND 6.1979−0.458433726 LYMPHOID TISSUE PC14 LUNG 6.2003 −0.455914571 KMS11HAEMATOPOIETIC AND 6.2008 −0.455389747 LYMPHOID TISSUE DMS53 LUNG 6.2061−0.449826613 SNU1214 UPPER AERODIGESTIVE 6.2071 −0.448776965 TRACT GOS3CENTRAL NERVOUS SYSTEM 6.2076 −0.448252141 TE8 OESOPHAGUS 6.2119−0.443738655 ECGI10 OESOPHAGUS 6.2151 −0.440379781 KO52 HAEMATOPOIETICAND 6.2174 −0.437965591 LYMPHOID TISSUE NCIH1793 LUNG 6.2189−0.436391119 NB4 HAEMATOPOIETIC AND 6.219 −0.436286155 LYMPHOID TISSUENCIH1105 LUNG 6.2191 −0.43618119 OCILY10 HAEMATOPOIETIC AND 6.222−0.433137211 LYMPHOID TISSUE NCIH69 LUNG 6.2243 −0.430723021 A673 BONE6.2304 −0.424320168 HCC4006 LUNG 6.2335 −0.42106626 SCC9 UPPERAERODIGESTIVE 6.2351 −0.419386823 TRACT OAW28 OVARY 6.2381 −0.416237879BXPC3 PANCREAS 6.2387 −0.415608091 ISTMES1 PLEURA 6.2389 −0.415398161SKMM2 HAEMATOPOIETIC AND 6.2396 −0.414663408 LYMPHOID TISSUE NCIN87STOMACH 6.24 −0.414243548 T98G CENTRAL NERVOUS SYSTEM 6.2412−0.412983971 GP2D LARGE INTESTINE 6.2536 −0.399968337 FTC238 THYROID6.2564 −0.397029323 KMS27 HAEMATOPOIETIC AND 6.2607 −0.392515837LYMPHOID TISSUE SNU201 CENTRAL NERVOUS SYSTEM 6.2618 −0.391361224 BC3CURINARY TRACT 6.266 −0.386952703 RS411 HAEMATOPOIETIC AND 6.2689−0.383908724 LYMPHOID TISSUE TALL1 HAEMATOPOIETIC AND 6.2742 −0.37834559LYMPHOID TISSUE RT4 URINARY TRACT 6.2742 −0.37834559 SKOV3 OVARY 6.2773−0.375091681 RERFLCAD2 LUNG 6.2783 −0.374042033 KHM1B HAEMATOPOIETIC AND6.2859 −0.366064709 LYMPHOID TISSUE KASUMI2 HAEMATOPOIETIC AND 6.2904−0.361341294 LYMPHOID TISSUE MOLT16 HAEMATOPOIETIC AND 6.2966−0.354833477 LYMPHOID TISSUE NUDUL1 HAEMATOPOIETIC AND 6.2966−0.354833477 LYMPHOID TISSUE KMS18 HAEMATOPOIETIC AND 6.2973−0.354098723 LYMPHOID TISSUE MDAMB175VII BREAST 6.2981 −0.353259005 RMGIOVARY 6.3019 −0.349270343 KIJK HAEMATOPOIETIC AND 6.305 −0.346016434LYMPHOID TISSUE OCIAML5 HAEMATOPOIETIC AND 6.3062 −0.344756857 LYMPHOIDTISSUE KMRC20 KIDNEY 6.3063 −0.344651892 LU65 LUNG 6.3082 −0.342657561JIMT1 BREAST 6.3087 −0.342132737 SNU8 OVARY 6.3089 −0.341922807 KALS1CENTRAL NERVOUS SYSTEM 6.3098 −0.340978124 SCABER URINARY TRACT 6.322−0.32817242 OVMANA OVARY 6.3268 −0.32313411 TUHR10TKB KIDNEY 6.3302−0.319565307 SUPM2 HAEMATOPOIETIC AND 6.3314 −0.318305729 LYMPHOIDTISSUE JMSU1 URINARY TRACT 6.3317 −0.317990835 NCIH446 LUNG 6.3331−0.316521328 COV434 OVARY 6.3341 −0.31547168 HCC38 BREAST 6.3361−0.313372384 KMRC2 KIDNEY 6.3393 −0.310013511 SNU478 BILIARY TRACT6.3432 −0.305919884 SUDHL1 HAEMATOPOIETIC AND 6.3444 −0.304660306LYMPHOID TISSUE CMLT1 HAEMATOPOIETIC AND 6.3494 −0.299412067 LYMPHOIDTISSUE UACC257 SKIN 6.3508 −0.29794256 NCIH1339 LUNG 6.3509 −0.297837595M07E HAEMATOPOIETIC AND 6.3511 −0.297627665 LYMPHOID TISSUE KMRC3 KIDNEY6.3514 −0.297312771 NCIH1693 LUNG 6.3603 −0.287970905 MM1SHAEMATOPOIETIC AND 6.3604 −0.28786594 LYMPHOID TISSUE HCC1143 BREAST6.3611 −0.287131186 KATOIII STOMACH 6.3642 −0.283877278 MDAMB453 BREAST6.3691 −0.278734003 J82 URINARY TRACT 6.3718 −0.275899954 CAL27 UPPERAERODIGESTIVE 6.3725 −0.2751652 TRACT HS766T PANCREAS 6.3727−0.274955271 HCT8 LARGE INTESTINE 6.3733 −0.274325482 NCIH1581 LUNG6.3747 −0.272855975 REH HAEMATOPOIETIC AND 6.3759 −0.271596397 LYMPHOIDTISSUE MPP89 PLEURA 6.3817 −0.265508439 SNU761 LIVER 6.3819 −0.26529851RH30 SOFT TISSUE 6.3841 −0.262989284 KURAMOCHI OVARY 6.3842 −0.26288432HS936T SKIN 6.385 −0.262044601 HCC15 LUNG 6.3861 −0.260889989 F36PHAEMATOPOIETIC AND 6.388 −0.258895657 LYMPHOID TISSUE PANC0504 PANCREAS6.3894 −0.25742615 NOMO1 HAEMATOPOIETIC AND 6.3925 −0.254172242 LYMPHOIDTISSUE SKUT1 SOFT TISSUE 6.3987 −0.247664425 CCK81 LARGE INTESTINE6.4043 −0.241786397 NCIH211 LUNG 6.4058 −0.240211925 NH6 AUTONOMICGANGLIA 6.4066 −0.239372206 BECKER CENTRAL NERVOUS SYSTEM 6.4161−0.229400551 NCIH1869 LUNG 6.4177 −0.227721114 ASPC1 PANCREAS 6.4186−0.226776431 VMCUB1 URINARY TRACT 6.4199 −0.225411889 SNU398 LIVER6.4206 −0.224677136 THP1 HAEMATOPOIETIC AND 6.4214 −0.223837417 LYMPHOIDTISSUE HS611T HAEMATOPOIETIC AND 6.4224 −0.222787769 LYMPHOID TISSUEONS76 CENTRAL NERVOUS SYSTEM 6.4253 −0.21974379 LOVO LARGE INTESTINE6.4266 −0.218379248 GMS10 CENTRAL NERVOUS SYSTEM 6.4313 −0.213445903 RKOLARGE INTESTINE 6.4316 −0.213131009 ZR7530 BREAST 6.4339 −0.210716818FU97 STOMACH 6.4421 −0.202109705 OCILY3 HAEMATOPOIETIC AND 6.4442−0.199905445 LYMPHOID TISSUE BV173 HAEMATOPOIETIC AND 6.4448−0.199275656 LYMPHOID TISSUE NCIH1568 LUNG 6.4489 −0.1949721 NCIH1155LUNG 6.4497 −0.194132381 JURKAT HAEMATOPOIETIC AND 6.4524 −0.191298332LYMPHOID TISSUE CW2 LARGE INTESTINE 6.4567 −0.186784846 RD SOFT TISSUE6.4567 −0.186784846 RERFLCAI LUNG 6.4571 −0.186364987 YD10B UPPERAERODIGESTIVE 6.4579 −0.185525268 TRACT SF295 CENTRAL NERVOUS SYSTEM6.4581 −0.185315339 JJN3 HAEMATOPOIETIC AND 6.4585 −0.18489548 LYMPHOIDTISSUE EB1 HAEMATOPOIETIC AND 6.4633 −0.17985717 LYMPHOID TISSUE KNS60CENTRAL NERVOUS SYSTEM 6.4642 −0.178912487 X697 HAEMATOPOIETIC AND6.4674 −0.175553613 LYMPHOID TISSUE TOV21G OVARY 6.4695 −0.173349353JHH5 LIVER 6.4703 −0.172509634 OVTOKO OVARY 6.4718 −0.170935162 WM1799SKIN 6.4744 −0.168206078 PL21 HAEMATOPOIETIC AND 6.4754 −0.16715643LYMPHOID TISSUE CA46 HAEMATOPOIETIC AND 6.4772 −0.165267064 LYMPHOIDTISSUE PATU8988S PANCREAS 6.479 −0.163377697 HCC44 LUNG 6.4794−0.162957838 KARPAS299 HAEMATOPOIETIC AND 6.4827 −0.159494 LYMPHOIDTISSUE PANC0327 PANCREAS 6.4856 −0.156450021 YD8 UPPER AERODIGESTIVE6.4856 −0.156450021 TRACT GDM1 HAEMATOPOIETIC AND 6.4875 −0.15445569LYMPHOID TISSUE IM95 STOMACH 6.4877 −0.154245761 HCT15 LARGE INTESTINE6.4918 −0.149942204 WM793 SKIN 6.4939 −0.147737944 SHP77 LUNG 6.5008−0.140495373 X8MGBA CENTRAL NERVOUS SYSTEM 6.5012 −0.140075514 OUMS23LARGE INTESTINE 6.5015 −0.139760619 SW1116 LARGE INTESTINE 6.5032−0.137976218 NCIH1703 LUNG 6.5035 −0.137661324 HLF LIVER 6.5042−0.13692657 REC1 HAEMATOPOIETIC AND 6.5051 −0.135981887 LYMPHOID TISSUEML1 THYROID 6.5066 −0.134407415 HOS BONE 6.5069 −0.134092521 SW837 LARGEINTESTINE 6.5072 −0.133777626 EHEB HAEMATOPOIETIC AND 6.5124−0.128319457 LYMPHOID TISSUE HUH28 BILIARY TRACT 6.5145 −0.126115197MDAMB157 BREAST 6.5173 −0.123176182 CHP212 AUTONOMIC GANGLIA 6.5178−0.122651359 RMUGS OVARY 6.52 −0.120342133 NCIH2106 LUNG 6.5249−0.115198858 SKLMS1 SOFT TISSUE 6.5254 −0.114674034 X647V URINARY TRACT6.5257 −0.11435914 HS294T SKIN 6.5258 −0.114254175 CHAGOK1 LUNG 6.5292−0.110685372 NCIH2228 LUNG 6.5304 −0.109425795 MHHCALL3 HAEMATOPOIETICAND 6.5324 −0.107326499 LYMPHOID TISSUE TE6 OESOPHAGUS 6.5328−0.10690664 MHHES1 BONE 6.5353 −0.10428252 X42MGBA CENTRAL NERVOUSSYSTEM 6.5397 −0.099664069 SH10TC STOMACH 6.5448 −0.094310865 HCC202BREAST 6.5484 −0.090532132 ACHN KIDNEY 6.5518 −0.08696333 SCC25 UPPERAERODIGESTIVE 6.5527 −0.086018646 TRACT PANC0403 PANCREAS 6.5578−0.080665442 A2780 OVARY 6.5613 −0.076991674 EBC1 LUNG 6.5617−0.076571815 SW620 LARGE INTESTINE 6.5658 −0.072268259 SKMEL31 SKIN6.5659 −0.072163294 PK45H PANCREAS 6.5666 −0.07142854 NCIH2030 LUNG6.5688 −0.069119315 SKMES1 LUNG 6.5724 −0.065340583 NAMALWAHAEMATOPOIETIC AND 6.5738 −0.063871075 LYMPHOID TISSUE CAL12T LUNG6.5741 −0.063556181 HPBALL HAEMATOPOIETIC AND 6.5743 −0.063346251LYMPHOID TISSUE HT1080 SOFT TISSUE 6.5745 −0.063136322 OE33 OESOPHAGUS6.5749 −0.062716463 SR786 HAEMATOPOIETIC AND 6.5751 −0.062506533LYMPHOID TISSUE NCIH929 HAEMATOPOIETIC AND 6.5755 −0.062086674 LYMPHOIDTISSUE OVCAR4 OVARY 6.5755 −0.062086674 T47D BREAST 6.5764 −0.061141991HCC1937 BREAST 6.5773 −0.060197308 SKHEP1 LIVER 6.5773 −0.060197308KMS26 HAEMATOPOIETIC AND 6.5778 −0.059672484 LYMPHOID TISSUE SNU1066UPPER AERODIGESTIVE 6.5779 −0.059567519 TRACT SUPHD1 HAEMATOPOIETIC AND6.5802 −0.057153329 LYMPHOID TISSUE L428 HAEMATOPOIETIC AND 6.5828−0.054424244 LYMPHOID TISSUE PLCPRF5 LIVER 6.584 −0.053164667 MSTO211HPLEURA 6.5871 −0.049910758 GA10 HAEMATOPOIETIC AND 6.59 −0.046866779LYMPHOID TISSUE HSC2 UPPER AERODIGESTIVE 6.59 −0.046866779 TRACT MKN74STOMACH 6.5911 −0.045712167 TOLEDO HAEMATOPOIETIC AND 6.5926−0.044137695 LYMPHOID TISSUE KARPAS620 HAEMATOPOIETIC AND 6.5931−0.043612871 LYMPHOID TISSUE CALU6 LUNG 6.5932 −0.043507906 SNU1196BILIARY TRACT 6.5947 −0.041933434 HGC27 STOMACH 6.595 −0.04161854NCIH716 LARGE INTESTINE 6.5964 −0.040149033 HDMYZ HAEMATOPOIETIC AND6.5974 −0.039099385 LYMPHOID TISSUE A3KAW HAEMATOPOIETIC AND 6.6031−0.033116392 LYMPHOID TISSUE SNGM ENDOMETRIUM 6.6038 −0.032381638 CAL851BREAST 6.6074 −0.028602906 JHUEM2 ENDOMETRIUM 6.608 −0.027973117 LN18CENTRAL NERVOUS SYSTEM 6.6106 −0.025244032 VMRCRCZ KIDNEY 6.6107−0.025139067 TE10 OESOPHAGUS 6.6127 −0.023039772 CAKI2 KIDNEY 6.614−0.021675229 PK1 PANCREAS 6.6156 −0.019995793 TE1 OESOPHAGUS 6.6158−0.019785863 IGR39 SKIN 6.6163 −0.019261039 NCIH1781 LUNG 6.6169−0.01863125 A253 SALIVARY GLAND 6.6238 −0.01138868 NCIH727 LUNG 6.6253−0.009814208 G361 SKIN 6.6284 −0.006560299 TYKNU OVARY 6.6296−0.005300722 SNU1041 UPPER AERODIGESTIVE 6.6307 −0.004146109 TRACT JL1PLEURA 6.6309 −0.00393618 SNU283 LARGE INTESTINE 6.6315 −0.003306391HCT116 LARGE INTESTINE 6.632 −0.002781567 LS1034 LARGE INTESTINE 6.6323−0.002466673 EFO21 OVARY 6.633 −0.001731919 DMS114 LUNG 6.6335−0.001207095 SNU1077 ENDOMETRIUM 6.6342 −0.000472342 DAOY CENTRALNERVOUS SYSTEM 6.6343 −0.000367377 NCIH2342 LUNG 6.6346 −5.24824E−05MOLP8 HAEMATOPOIETIC AND 6.6347 5.24824E−05 LYMPHOID TISSUE BHT101THYROID 6.6351 0.000472342 TE5 OESOPHAGUS 6.6355 0.000892201 PSN1PANCREAS 6.6403 0.005930511 NCIH2170 LUNG 6.6424 0.008134771 RCHACVHAEMATOPOIETIC AND 6.6426 0.008344701 LYMPHOID TISSUE HUH6 LIVER 6.64370.009499314 NCIH838 LUNG 6.6448 0.010653926 YAPC PANCREAS 6.64850.014537624 KYSE450 OESOPHAGUS 6.6505 0.016636919 RERFLCMS LUNG 6.65120.017371673 OVISE OVARY 6.6514 0.017581603 HT55 LARGE INTESTINE 6.65540.021780194 SNU899 UPPER AERODIGESTIVE 6.662 0.02870787 TRACT NCIH226LUNG 6.6624 0.02912773 X639V URINARY TRACT 6.6635 0.030282342 TE14OESOPHAGUS 6.6652 0.032066744 MKN45 STOMACH 6.6662 0.033116392 UMUC3URINARY TRACT 6.6662 0.033116392 HEC6 ENDOMETRIUM 6.6667 0.033641216X253JBV URINARY TRACT 6.6694 0.036475265 SKMEL24 SKIN 6.6712 0.038364631VMRCLCD LUNG 6.6718 0.03899442 DLD1 LARGE INTESTINE 6.6751 0.042458258ECC12 STOMACH 6.6785 0.046027061 WSUDLCL2 HAEMATOPOIETIC AND 6.68010.047706498 LYMPHOID TISSUE PFEIFFER HAEMATOPOIETIC AND 6.68040.048021392 LYMPHOID TISSUE NCIH2087 LUNG 6.6806 0.048231322 NCIH2029LUNG 6.6826 0.050330617 SJSA1 BONE 6.6844 0.052219984 A172 CENTRALNERVOUS SYSTEM 6.6858 0.053689491 SNU1033 LARGE INTESTINE 6.68730.055263963 TM31 CENTRAL NERVOUS SYSTEM 6.6885 0.05652354 X2313287STOMACH 6.6886 0.056628505 SQ1 LUNG 6.6945 0.062821428 SUPT11HAEMATOPOIETIC AND 6.695 0.063346251 LYMPHOID TISSUE NCIH2023 LUNG6.6954 0.063766111 HCC1569 BREAST 6.6976 0.066075336 TT2609C02 THYROID6.7014 0.070063998 SW1990 PANCREAS 6.7019 0.070588822 OVSAHO OVARY6.7028 0.071533505 NCIH841 LUNG 6.7036 0.072373224 ME1 HAEMATOPOIETICAND 6.7039 0.072688118 LYMPHOID TISSUE COLO205 LARGE INTESTINE 6.70520.07405266 TCCSUP URINARY TRACT 6.7056 0.074472519 TE11 OESOPHAGUS6.7063 0.075207273 TE4 OESOPHAGUS 6.707 0.075942026 NCIH1694 LUNG 6.70950.078566146 KP4 PANCREAS 6.7102 0.0793009 CL11 LARGE INTESTINE 6.7110.080140618 NCIH596 LUNG 6.7123 0.08150516 OCIAML3 HAEMATOPOIETIC AND6.7152 0.084549139 LYMPHOID TISSUE KMH2 HAEMATOPOIETIC AND 6.71550.084864034 LYMPHOID TISSUE PK59 PANCREAS 6.7163 0.085703752 HDLM2HAEMATOPOIETIC AND 6.7172 0.086648435 LYMPHOID TISSUE ES2 OVARY 6.71830.087803048 SKNDZ AUTONOMIC GANGLIA 6.7192 0.088747731 NCIH650 LUNG6.7194 0.088957661 CAL62 THYROID 6.721 0.090637097 MDAMB231 BREAST6.7222 0.091896675 HARA LUNG 6.7238 0.093576111 MFE319 ENDOMETRIUM6.7242 0.093995971 LCLC103H LUNG 6.7269 0.09683002 OE19 OESOPHAGUS6.7273 0.097249879 HT144 SKIN 6.7297 0.099769034 HEC251 ENDOMETRIUM6.7301 0.100188893 A4FUK HAEMATOPOIETIC AND 6.7317 0.10186833 LYMPHOIDTISSUE K562 HAEMATOPOIETIC AND 6.7319 0.102078259 LYMPHOID TISSUE HEC59ENDOMETRIUM 6.7321 0.102288189 NCIH1341 LUNG 6.7337 0.103967626 A204SOFT TISSUE 6.7338 0.10407259 OV7 OVARY 6.7346 0.104912309 OV90 OVARY6.7381 0.108586076 HCC827 LUNG 6.7384 0.108900971 DU4475 BREAST 6.7420.112679703 SKMEL1 SKIN 6.742 0.112679703 KYSE70 OESOPHAGUS 6.74280.113519422 CHP126 AUTONOMIC GANGLIA 6.7459 0.11677333 DETROIT562 UPPERAERODIGESTIVE 6.7465 0.117403119 TRACT CMK HAEMATOPOIETIC AND 6.74830.119292485 LYMPHOID TISSUE X769P KIDNEY 6.7486 0.11960738 DELHAEMATOPOIETIC AND 6.7494 0.120447098 LYMPHOID TISSUE PANC0813 PANCREAS6.751 0.122126535 COLO201 LARGE INTESTINE 6.752 0.123176182 SKNMC BONE6.7533 0.124540725 CALU3 LUNG 6.7536 0.124855619 SNU1076 UPPERAERODIGESTIVE 6.7574 0.128844281 TRACT HCC78 LUNG 6.7625 0.134197486ESS1 ENDOMETRIUM 6.7626 0.13430245 NCIH1755 LUNG 6.771 0.143119493HPAFII PANCREAS 6.7751 0.147423049 CAKI1 KIDNEY 6.7755 0.147842908COLO783 SKIN 6.778 0.150467028 NCIH2405 LUNG 6.7785 0.150991852 KNS81CENTRAL NERVOUS SYSTEM 6.7793 0.15183157 HCC95 LUNG 6.7794 0.151936535HL60 HAEMATOPOIETIC AND 6.7796 0.152146465 LYMPHOID TISSUE FADU UPPERAERODIGESTIVE 6.7809 0.153511007 TRACT TE617T SOFT TISSUE 6.7820.15466562 KMBC2 URINARY TRACT 6.7837 0.156450021 HCC1171 LUNG 6.78380.156554986 CAPAN1 PANCREAS 6.786 0.158864211 CORL88 LUNG 6.79150.164637275 PECAPJ49 UPPER AERODIGESTIVE 6.7927 0.165896852 TRACT SF126CENTRAL NERVOUS SYSTEM 6.7933 0.166526641 GSS STOMACH 6.794 0.167261395U87MG CENTRAL NERVOUS SYSTEM 6.7949 0.168206078 HEYA8 OVARY 6.79720.170620268 HT1376 URINARY TRACT 6.7994 0.172929493 COLO792 SKIN 6.79970.173244388 SKMEL2 SKIN 6.8019 0.175553613 NCIH460 LUNG 6.80480.178597592 KU1919 URINARY TRACT 6.8061 0.179962134 SNU407 LARGEINTESTINE 6.8062 0.180067099 KU812 HAEMATOPOIETIC AND 6.8063 0.180172064LYMPHOID TISSUE NCIH747 LARGE INTESTINE 6.8075 0.181431642 A101D SKIN6.8089 0.182901149 PATU8988T PANCREAS 6.8099 0.183950797 HS895T SKIN6.8118 0.185945128 HMC18 BREAST 6.8147 0.188989107 X253J URINARY TRACT6.8153 0.189618895 TE9 OESOPHAGUS 6.8154 0.18972386 LS123 LARGEINTESTINE 6.8175 0.191928121 MCAS OVARY 6.8199 0.194447276 SW403 LARGEINTESTINE 6.8208 0.195391959 MDST8 LARGE INTESTINE 6.8209 0.195496924RCM1 LARGE INTESTINE 6.8231 0.197806149 NCIH1650 LUNG 6.825 0.19980048RPMI8226 HAEMATOPOIETIC AND 6.8256 0.200430269 LYMPHOID TISSUE SUDHL8HAEMATOPOIETIC AND 6.8258 0.200640198 LYMPHOID TISSUE HEPG2 LIVER 6.82740.202319635 HT115 LARGE INTESTINE 6.8303 0.205363614 KYSE520 OESOPHAGUS6.8305 0.205573544 ISHIKAWAHERAKLIO02ER ENDOMETRIUM 6.8313 0.206413262RT112 URINARY TRACT 6.8313 0.206413262 SNU308 BILIARY TRACT 6.83140.206518227 HCC1806 BREAST 6.8314 0.206518227 NCIH2085 LUNG 6.83170.206833121 EFO27 OVARY 6.832 0.207148015 NCIH2052 PLEURA 6.83210.20725298 HSC4 UPPER AERODIGESTIVE 6.8327 0.207882769 TRACT KYSE140OESOPHAGUS 6.836 0.211346607 LC1SQSF LUNG 6.8361 0.211451572 KMRC1KIDNEY 6.8362 0.211556537 HUPT3 PANCREAS 6.837 0.212396255 NCIH1838 LUNG6.8375 0.212921079 T24 URINARY TRACT 6.8383 0.213760797 WM115 SKIN6.8396 0.21512534 KASUMI1 HAEMATOPOIETIC AND 6.8439 0.219638826 LYMPHOIDTISSUE GAMG CENTRAL NERVOUS SYSTEM 6.8471 0.222997699 SBC5 LUNG 6.84940.225411889 WM2664 SKIN 6.8521 0.228245938 D283MED CENTRAL NERVOUSSYSTEM 6.857 0.233389213 MIAPACA2 PANCREAS 6.8607 0.23727291 BL70HAEMATOPOIETIC AND 6.8619 0.238532488 LYMPHOID TISSUE NCIH1623 LUNG6.862 0.238637453 BHY UPPER AERODIGESTIVE 6.8627 0.239372206 TRACTOVCAR8 OVARY 6.8637 0.240421854 SNU840 OVARY 6.8651 0.241891361 CFPAC1PANCREAS 6.8671 0.243990657 HS944T SKIN 6.8697 0.246719742 LK2 LUNG6.8724 0.249553791 JHH1 LIVER 6.8737 0.250918333 OVKATE OVARY 6.87420.251443157 T84 LARGE INTESTINE 6.8791 0.256586432 SW1573 LUNG 6.88130.258895657 KYSE30 OESOPHAGUS 6.8825 0.260155235 DANG PANCREAS 6.88250.260155235 SU8686 PANCREAS 6.8851 0.26288432 YD15 SALIVARY GLAND 6.88580.263619073 COLO680N OESOPHAGUS 6.8864 0.264248862 SUDHL6 HAEMATOPOIETICAND 6.887 0.264878651 LYMPHOID TISSUE SNU626 CENTRAL NERVOUS SYSTEM6.8886 0.266558087 SNU1105 CENTRAL NERVOUS SYSTEM 6.8918 0.269916961BT20 BREAST 6.8931 0.271281503 FTC133 THYROID 6.8949 0.273170869P12ICHIKAWA HAEMATOPOIETIC AND 6.8951 0.273380799 LYMPHOID TISSUENCIH292 LUNG 6.8954 0.273695693 JHH2 LIVER 6.9004 0.278943933 RCC10RGBKIDNEY 6.9009 0.279468757 JHOC5 OVARY 6.9036 0.282302806 X786O KIDNEY6.9057 0.284507067 AN3CA ENDOMETRIUM 6.9081 0.287026222 KP3 PANCREAS6.909 0.287970905 HEC151 ENDOMETRIUM 6.9099 0.288915588 KE39 STOMACH6.9103 0.289335447 HS822T BONE 6.9115 0.290595024 A375 SKIN 6.91170.290804954 MORCPR LUNG 6.9126 0.291749637 C2BBE1 LARGE INTESTINE 6.91440.293639003 NCIH2452 PLEURA 6.9169 0.296263123 TCCPAN2 PANCREAS 6.91840.297837595 VMRCRCW KIDNEY 6.9222 0.301826257 NCIH810 LUNG 6.92220.301826257 PC3 PROSTATE 6.9226 0.302246116 MDAMB435S SKIN 6.92270.302351081 NCIH322 LUNG 6.9254 0.30518513 MOLP2 HAEMATOPOIETIC AND6.928 0.307914215 LYMPHOID TISSUE HCC366 LUNG 6.9295 0.309488687 KELLYAUTONOMIC GANGLIA 6.9352 0.31547168 AGS STOMACH 6.9378 0.318200764MDAMB468 BREAST 6.9388 0.319250412 SNUC5 LARGE INTESTINE 6.9390.319460342 HCC1195 LUNG 6.941 0.321559638 NB1 AUTONOMIC GANGLIA 6.94660.327437666 NCIH2126 LUNG 6.9473 0.32817242 HT HAEMATOPOIETIC AND 6.94760.328487314 LYMPHOID TISSUE SW48 LARGE INTESTINE 6.9505 0.331531293 QGP1PANCREAS 6.9517 0.33279087 NUGC3 STOMACH 6.9527 0.333840518 SNU719STOMACH 6.9544 0.33562492 SKES1 BONE 6.9576 0.338983793 OVK18 OVARY6.9579 0.339298688 HEC1B ENDOMETRIUM 6.9583 0.339718547 KLE ENDOMETRIUM6.9584 0.339823511 HEC50B ENDOMETRIUM 6.9622 0.343812174 TF1HAEMATOPOIETIC AND 6.9682 0.350110061 LYMPHOID TISSUE AM38 CENTRALNERVOUS SYSTEM 6.9715 0.353573899 HCC1954 BREAST 6.9728 0.354938441MELHO SKIN 6.9769 0.359241998 EN ENDOMETRIUM 6.9773 0.359661857 HCC2108LUNG 6.9789 0.361341294 X22RV1 PROSTATE 6.9813 0.363860449 PATU8902PANCREAS 6.9874 0.370263301 LN229 CENTRAL NERVOUS SYSTEM 6.98830.371207984 GI1 CENTRAL NERVOUS SYSTEM 6.9897 0.372677491 SNU213PANCREAS 6.9923 0.375406576 COLO684 ENDOMETRIUM 6.993 0.376141329 SNU738CENTRAL NERVOUS SYSTEM 6.9945 0.377715801 JK1 HAEMATOPOIETIC AND 6.99660.379920062 LYMPHOID TISSUE KYSE510 OESOPHAGUS 6.9987 0.382124322NCIH1299 LUNG 6.9991 0.382544181 IGROV1 OVARY 7.0026 0.386217949ACCMESO1 PLEURA 7.0033 0.386952703 BICR16 UPPER AERODIGESTIVE 7.00710.390941365 TRACT HCC2279 LUNG 7.0072 0.39104633 PANC1 PANCREAS 7.00960.393565485 CCFSTTG1 CENTRAL NERVOUS SYSTEM 7.0119 0.395979675 SNU668STOMACH 7.0126 0.396714428 SW1271 LUNG 7.0143 0.39849883 SUDHL4HAEMATOPOIETIC AND 7.0162 0.400493161 LYMPHOID TISSUE GCT SOFT TISSUE7.0174 0.401752738 TT THYROID 7.0183 0.402697421 DMS454 LUNG 7.0190.403432175 LS180 LARGE INTESTINE 7.0225 0.407105943 SNU182 LIVER 7.02520.409939992 KNS62 LUNG 7.0253 0.410044957 OC314 OVARY 7.0273 0.412144253RH41 SOFT TISSUE 7.0285 0.41340383 NCIH1373 LUNG 7.0318 0.416867668 BENLUNG 7.0341 0.419281858 MESSA SOFT TISSUE 7.0401 0.425579746 HEC1AENDOMETRIUM 7.0465 0.432297493 L363 HAEMATOPOIETIC AND 7.04730.433137211 LYMPHOID TISSUE CAL29 URINARY TRACT 7.0497 0.435656366 RAJIHAEMATOPOIETIC AND 7.0524 0.438490415 LYMPHOID TISSUE ZR751 BREAST 7.0540.440169852 KYSE180 OESOPHAGUS 7.0541 0.440274817 LOXIMVI SKIN 7.0580.444368444 YD38 UPPER AERODIGESTIVE 7.06 0.446467739 TRACT SNU410PANCREAS 7.0646 0.45129612 NCIH2291 LUNG 7.0654 0.452135838 PANC0203PANCREAS 7.0662 0.452975556 NCIH1792 LUNG 7.0701 0.457069183 SW1088CENTRAL NERVOUS SYSTEM 7.0786 0.46599119 SKMEL30 SKIN 7.079 0.46641105KM12 LARGE INTESTINE 7.0792 0.466620979 HEC108 ENDOMETRIUM 7.08040.467880557 NCIH526 LUNG 7.0825 0.470084817 NCIH661 LUNG 7.08320.470819571 KYSE150 OESOPHAGUS 7.0859 0.47365362 TUHR4TKB KIDNEY 7.08610.47386355 U251MG CENTRAL NERVOUS SYSTEM 7.091 0.479006825 MKN1 STOMACH7.0915 0.479531649 DMS273 LUNG 7.0958 0.484045135 HS683 CENTRAL NERVOUSSYSTEM 7.0975 0.485829536 HS746T STOMACH 7.1012 0.489713233 OAW42 OVARY7.1038 0.492442318 KYO1 HAEMATOPOIETIC AND 7.1048 0.493491966 LYMPHOIDTISSUE HS688AT SKIN 7.1049 0.493596931 SIGM5 HAEMATOPOIETIC AND 7.10770.496535945 LYMPHOID TISSUE HUCCT1 BILIARY TRACT 7.1094 0.498320346HS819T BONE 7.1097 0.498635241 HCC1588 LUNG 7.1149 0.50409341 KPL1BREAST 7.1178 0.507137389 KE97 HAEMATOPOIETIC AND 7.1187 0.508082072LYMPHOID TISSUE HCC2218 BREAST 7.1208 0.510286332 OCIM1 HAEMATOPOIETICAND 7.1253 0.515009748 LYMPHOID TISSUE NCIH441 LUNG 7.1284 0.518263657NCIH1092 LUNG 7.139 0.529389924 SKMEL28 SKIN 7.1392 0.529599854 HPACPANCREAS 7.1394 0.529809784 SAOS2 BONE 7.1406 0.531069361 RL952ENDOMETRIUM 7.1432 0.533798446 SKNAS AUTONOMIC GANGLIA 7.145 0.535687812CAL148 BREAST 7.1477 0.538521861 DMS79 LUNG 7.1572 0.548493516 EFE184ENDOMETRIUM 7.1614 0.552902038 SUPT1 HAEMATOPOIETIC AND 7.1670.558780066 LYMPHOID TISSUE NMCG1 CENTRAL NERVOUS SYSTEM 7.17460.56675739 NCIH358 LUNG 7.1753 0.567492144 TE441T SOFT TISSUE 7.17720.569486475 MELJUSO SKIN 7.1877 0.580507778 IPC298 SKIN 7.19840.59173901 SW1353 BONE 7.1985 0.591843975 CAL33 UPPER AERODIGESTIVE7.2038 0.597407109 TRACT SNU489 CENTRAL NERVOUS SYSTEM 7.20560.599296475 LCLC97TM1 LUNG 7.2086 0.602445419 BICR56 UPPER AERODIGESTIVE7.2108 0.604754644 TRACT NCIH508 LARGE INTESTINE 7.2176 0.61189225 HSC3UPPER AERODIGESTIVE 7.2237 0.618295103 TRACT SNU878 LIVER 7.22380.618400067 CAMA1 BREAST 7.2254 0.620079504 LS411N LARGE INTESTINE7.2279 0.622703624 YKG1 CENTRAL NERVOUS SYSTEM 7.2376 0.632885208 JHH6LIVER 7.2377 0.632990173 KG1C CENTRAL NERVOUS SYSTEM 7.238 0.633305068BT474 BREAST 7.2422 0.637713589 SNU1079 BILIARY TRACT 7.2463 0.642017145KARPAS422 HAEMATOPOIETIC AND 7.2487 0.6445363 LYMPHOID TISSUE HEC265ENDOMETRIUM 7.2509 0.646845526 NCIH2444 LUNG 7.2606 0.65702711 NUDHL1HAEMATOPOIETIC AND 7.2677 0.664479611 LYMPHOID TISSUE AMO1HAEMATOPOIETIC AND 7.2764 0.673611547 LYMPHOID TISSUE HCC1833 LUNG7.2887 0.686522217 SNUC4 LARGE INTESTINE 7.2927 0.690720808 HDQP1 BREAST7.2935 0.691560527 OV56 OVARY 7.2957 0.693869752 P3HR1 HAEMATOPOIETICAND 7.2973 0.695549189 LYMPHOID TISSUE NUGC4 STOMACH 7.2991 0.697438555U2OS BONE 7.3013 0.69974778 SNU886 LIVER 7.3032 0.701742112 NCIH28PLEURA 7.3081 0.706885386 SNU601 STOMACH 7.3091 0.707935034 ECC10STOMACH 7.3182 0.71748683 LS513 LARGE INTESTINE 7.3199 0.719271232CAL120 BREAST 7.32 0.719376196 SNU1040 LARGE INTESTINE 7.32880.728613098 NCIH2171 LUNG 7.3416 0.742048591 SUDHL5 HAEMATOPOIETIC AND7.3508 0.751705352 LYMPHOID TISSUE BFTC905 URINARY TRACT 7.35140.752335141 HT29 LARGE INTESTINE 7.364 0.765560705 RPMI7951 SKIN 7.3750.777106832 AML193 HAEMATOPOIETIC AND 7.3753 0.777421726 LYMPHOID TISSUEMEC1 HAEMATOPOIETIC AND 7.376 0.778156479 LYMPHOID TISSUE HEP3B217 LIVER7.4062 0.809855846 SNU475 LIVER 7.4091 0.812899825 HUH1 LIVER 7.42980.834627537 HUPT4 PANCREAS 7.4555 0.861603488 IMR32 AUTONOMIC GANGLIA7.4593 0.865592151 NCIH889 LUNG 7.4952 0.903274511 HCC2935 LUNG 7.50840.917129863 MC116 HAEMATOPOIETIC AND 7.5146 0.92363768 LYMPHOID TISSUEX5637 URINARY TRACT 7.5183 0.927521377 SKM1 HAEMATOPOIETIC AND 7.52340.932874582 LYMPHOID TISSUE SKBR3 BREAST 7.5494 0.960165427 EM2HAEMATOPOIETIC AND 7.5755 0.987561238 LYMPHOID TISSUE RI1 HAEMATOPOIETICAND 7.5915 1.004355605 LYMPHOID TISSUE SIMA AUTONOMIC GANGLIA 7.60321.016636485 FUOV1 OVARY 7.6122 1.026083316 SNUC2A LARGE INTESTINE 7.61651.030596802 SNU61 LARGE INTESTINE 7.6228 1.037209584 CAPAN2 PANCREAS7.6273 1.041933 SNU216 STOMACH 7.6319 1.04676138 MOLM13 HAEMATOPOIETICAND 7.646 1.061561416 LYMPHOID TISSUE HUNS1 HAEMATOPOIETIC AND 7.66481.081294796 LYMPHOID TISSUE HCC1438 LUNG 7.7264 1.145953108 NCIH2196LUNG 7.7386 1.158758812 SNU466 CENTRAL NERVOUS SYSTEM 7.7589 1.180066665SUDHL10 HAEMATOPOIETIC AND 7.7977 1.220793004 LYMPHOID TISSUE SNU46UPPER AERODIGESTIVE 7.8035 1.226880962 TRACT CALU1 LUNG 7.81851.242625681 BFTC909 KIDNEY 7.9189 1.348010331 JVM3 HAEMATOPOIETIC AND7.961 1.392200508 LYMPHOID TISSUE MHHCALL4 HAEMATOPOIETIC AND 8.0311.465675862 LYMPHOID TISSUE JURLMK1 HAEMATOPOIETIC AND 8.11261.551327131 LYMPHOID TISSUE KE37 HAEMATOPOIETIC AND 8.1163 1.555210829LYMPHOID TISSUE S117 SOFT TISSUE 8.2668 1.713182839 KMS21BMHAEMATOPOIETIC AND 8.3309 1.780465271 LYMPHOID TISSUE KYM1 SOFT TISSUE8.4417 1.896766259 CORL95 LUNG 8.5762 2.037943903 MHHNB11 AUTONOMICGANGLIA 8.8255 2.299621128 MDAMB361 BREAST 9.2909 2.788127266

Example 2. Identification of PDE3A as a Putative Target of DNMDP

Given the potent cell-selective growth inhibition by6-(4-(diethylamino)-3-nitrophenyl)-5-methyl-4,5-dihydropyridazin-3(2H)-one(DNMDP), its mechanism of action was examined in more detail. Todetermine the molecular target of DNMDP, chemogenomic analysis wasperformed of the 766 tested cell lines, previously characterized formutations, copy number, and gene expression features as part of theCancer Cell Line Encyclopedia (CCLE, Barretina et al., 2012), to lookfor correlation between these genomic features and DNMDP sensitivity.Analysis of Pearson correlations between DNMDP sensitivity andexpression of individual genes across the cell line set showed a strongcorrelation with expression of the PDE3A gene, encodingphosphodiesterase 3A (FIG. 5A). The correlation between DNMDPsensitivity and PDE3A expression is not perfect (FIG. 18), and it ispossible that some errors are introduced due to the high-throughputnature of the cell line sensitivity characterization, as manualvalidation for all 766 cell lines was not logistically feasible.Mutation and copy number features, in contrast, did not correlate withDNMDP sensitivity. Conversely, of 480 compounds tested, DNMDPsensitivity was the closest correlate of PDE3A expression (FIG. 5B),indicating that cancer cell lines with high PDE3A expression were moredistinctly sensitive to DNMDP than to any other tested compound. Incontrast to the motivation of the initial screen, there was nocorrelation between TP53 mutation, or other measures of p53 function,and DNMDP sensitivity.

Given these results and the clear structural similarity of DNMDP toknown PDE3 inhibitors, e.g., levosimendan and siguazodan (FIGS. 6A-6C),biochemical analysis of DNMDP against 19 phosphodiesterases representing11 PDE super families was performed. At a concentration of 100 nM, DNMDPspecifically inhibited both PDE3A and PDE3B, weakly inhibited PDE10, andhad little or no detectable effect on other phosphodiesterases (Table2).

Because of the cellular correlation between PDE3A expression and DNMDPsensitivity, the in vitro inhibition of PDE3A and PDE3B by DNMDP, andthe structural similarity of DNMDP to known PDE3 inhibitors, it wasanalyzed whether all PDE3 inhibitors would exhibit a similar cytotoxicprofile to DNMDP. Surprisingly, there was almost no correlation betweenIC₅₀ for in vitro enzymatic PDE3A inhibition and HeLa cell cytotoxicityacross a series of tested compounds (FIG. 5C and FIGS. 7A and 7B).Indeed, the potent PDE3 inhibitor trequinsin (PDE3 IC₅₀=0.25 nM, Ruppertet al., Life Sci. 31, 2037-2043, 1982) did not affect HeLa cellviability in any detectable way. Despite their differential effects onHeLa cell viability, the non-cytotoxic PDE3 inhibitor trequinsin and thepotent cytotoxic compound DNMDP had similar effects on intracellularcAMP levels in forskolin-treated HeLa cells (FIGS. 8A and 8B). Thisresult indicates that inhibition of the cAMP and cGMP hydrolysisfunctions of PDE3A was not sufficient for the cytotoxic activity ofDNMDP.

TABLE 2 Results of phosphodiesterase inhibition reactions PDE % inh. #1% inh. #2 % inhibition PDE1A1 3 7 5 PDE1B −5 0 −2 PDE1C 2 9 5 PDE2A 6 108 PDE3A 95 95 95 PDE3B 98 97 97 PDE4A1A 14 18 16 PDE4B1 21 20 21 PDE4C110 14 12 PDE4D3 14 16 15 PDE4D7 19 20 20 PDE5A1 16 16 16 PDE7A 24 20 22PDE7B 5 11 8 PDE8A1 10 12 11 PDE9A2 0 5 2 PDE10A1 61 65 63 PDE10A2 67 7068 PDE11A 14 18 16

Example 3. Target Validation of PDE3A

The complex relationship between phosphodiesterase 3A (PDE3A) inhibitionand cell killing, in which6-(4-(diethylamino)-3-nitrophenyl)-5-methyl-4,5-dihydropyridazin-3(2H)-one(DNMDP) and some PDE3 inhibitors kill HeLa and other DNMDP-sensitivecells, whereas others PDE3 inhibitors do not affect cell viability,indicated several possible interpretations including: 1) the cytotoxicactivity might be PDE3-independent and due to action on a differentprotein though screening 234 kinases found no kinase inhibition by 10 μMDNMDP; 2) cytotoxic and non-cytotoxic PDE3 inhibitors might bind todifferent sites within the protein and exert distinct activities; or 3)the cytotoxic and non-cytotoxic PDE3 inhibitors might bind to the PDE3active sites but have different effects on the conformation and activityof the protein. This third possibility might be unexpected, butallosteric modulators of PDE4 have been shown to bind the PDE4 activesite and interact with upstream (UCR2), and downstream (CR3) regulatorydomains and thereby stabilize specific inactive conformations (Burgin etal., Nat Biotechnol 28, 63-70, 2010). Most importantly, PDE4 competitiveinhibitors and PDE4 allosteric modulators with similar IC₅₀s for cAMPhydrolysis in vitro had different cellular activities and safetyprofiles in animal studies (Burgin et al., Nat Biotechnol 28, 63-70,2010). To evaluate whether PDE inhibitors or other small moleculescompete with DNMDP, the PHARMAKON 1600 collection of 1600 bioactivecompounds (PHARMAKON 1600 is a unique collection of 1600 known drugsfrom US and International Pharmacopeia) was screened to identifycompounds that were able to rescue cell death induced by DNMDP. HeLacells were co-treated with 30 nM DNMDP (the EC₇₀ concentration) and 20μM of each bioactive compound. Cell viability after 48-hour treatmentwas assessed by ATP consumption as described earlier. The five mostpotent compounds that rescued cell death induced by DNMDP were all PDEinhibitors, and the three most potent compounds, levosimendan,milrinone, and cilostazol, were all selective PDE3 inhibitors (FIG. 9A).

In follow-up experiments, it was confirmed that cilostamide,levosimendan, milrinone, and several other non-cytotoxic selective PDE3inhibitors were able to rescue DNMDP cytotoxicity in a dose-dependentmanner (FIG. 9B). The most potent DNMDP competitor was trequinsin, withan “RC₅₀” (the concentration at which it achieved 50% rescue) of <1 nM;in contrast, PDE5 inhibitors such as sildenafil and vardenafil, as wellas the pan-PDE inhibitors idubulast and dipyridamole, were not effectivecompetitors up to 10 μM concentrations in this assay (FIG. 9B). Thisindicated that non-cytotoxic PDE3 inhibitors and DNMDP compete forbinding to the same molecular target that is mediating the cytotoxicphenotype.

To identify the molecular target of DNMDP, an affinity purification wasperformed using an (R)-des-nitro-DNMDP solid-phase tethered linkeranalogue (FIG. 10A) incubated with HeLa cell lysate. This linkeranalogue had the same DNMDP cytotoxicity rescue phenotype asnon-cytotoxic PDE3 inhibitors described above (FIG. 10B), indicatingthat it too bound to the same molecular target. It was competed for themolecular target by adding either an excess of trequinsin or separateenantiomers of DNMDP, where only the (R)-enantiomer was cytotoxic.Immunoblotting for PDE3A of the affinity purified material showed thatPDE3A indeed binds to the linker analogue. Binding of PDE3A to thelinker analogue was blocked by both trequinsin and (R)-DNMDP, but not bythe non-cytotoxic enantiomer (S)-DNMDP (FIG. 9C). Thus both trequinsinand (R)-DNMDP prevented the binding of PDE3A to the tethered DNMDPanalogue, and it was concluded that both molecules bind PDE3A directly.

Based on the observations that DNMDP-sensitive cells expressed highlevels of PDE3A, and that DNMDP competed with non-cytotoxic inhibitorsfor PDE3A binding, it was hypothesized that DNMDP mediated its cytotoxicphenotype through the interaction with PDE3A and that PDE3A abundancewas a direct cellular determinant of DNMDP sensitivity. To validate thishypothesis, the effect of reducing levels of PDE3A on the response toDNMDP was tested. A clustered regularly interspaced short palindromic(CRISPR)-associated CAS9 enzyme that was targeted with three guide RNAs(sgRNA) targeting three different sites in the PDE3A locus led tocomplete loss of PDE3A expression (Cong et al., Science 339, 819-823,2013) sgRNA2 and sgRNA3 almost completely reduced PDE3A protein levels,whereas sgRNA1 had a moderate effect on PDE3A expression (FIG. 11A).Importantly, both sgRNA2 and sgRNA3 led to significant rescue oftoxicity by an active cytotoxic DNMDP analog, 3 (FIGS. 11A and 11B andFIGS. 5A-5C). Both sgRNA2 and sgRNA3 led to significant rescue oftoxicity by DNMDP (FIG. 11C). Changes in proliferation rate ormorphology in HeLa cells with reduced PDE3A expression were notobserved, indicating that PDE3A was not required for cell survival. Inan independent approach using an siRNA smart-pool containing fourdifferent siRNAs targeting PDE3A, PDE3A expression was reduced in HeLacell line with a maximum efficiency of 70% between 24 and 72 hours aftertransfection. HeLa cells treated with siPDE3A had a higher EC₅₀ to aDNMDP analog compared to the control siRNA condition (FIGS. 12A and12B). Without being bound by theory it was concluded that DNMDPcytotoxicity requires PDE3A, and that DNMDP likely modulates thefunction of PDE3A.

Example 4. Determining the Mechanism of Action of DNMDP

The dependence of6-(4-(diethylamino)-3-nitrophenyl)-5-methyl-4,5-dihydropyridazin-3(2H)-one(DNMDP) cytotoxicity on phosphodiesterase 3A (PDE3A) protein abundanceindicated a possible mechanism similar to that recently observed forlenalidomide, which acts by a neomorphic or hypermorphic mechanism bystabilizing an interaction between cereblon and IKAROS Family ZincFinger 1 (IKZF1) and IKZF3 (Krönke et al., Science 343, 301-305, 2014;Lu et al., Science 343, 305-309, 2014). In addition, PDE4 allostericmodulators, but not competitive inhibitors, have been shown to bind andstabilize a “closed” protein conformation that has independently beenshown to uniquely bind the PDE4-partner protein DISC1 (Millar et al.,Science 310, 1187-1191, 2005). The protein complexes in which PDE3Aresides were characterized under normal conditions, and it was examinedhow these complexes change when PDE3A is bound to DNMDP or thenon-cytotoxic PDE3 inhibitor trequinsin. PDE3A and interacting proteinsfrom Hela cells were immunoprecipitated in the presence of DNMDP andtrequinsin followed by labeling with isobaric stable isotope tags forrelative abundance and quantitation by mass spectrometry (iTRAQ/MS, FIG.13A). PDE3A immunoprecipitates from HeLa cells were enriched formultiple protein phosphatase subunits including protein phosphatase 2subunits (PPP2CA, PPP2R1A, PPP2R1B, PPP2R2A, PPP2R2D), calcineurin(PPP3R1, PPP3CA, Beca et al., Circ. Res. 112, 289-297, 2013), 14-3-3(YWHAB, YWHAQ, YWHAG, YWHAZ, Pozuelo Rubio et al., Biochem. J. 392,163-172, 2005), and tubulin (TUBA1C, TUBA1B) family members (FIG. 13Band FIG. 14A). In addition, it was found that PDE3A and PDE3B reside inthe same protein complex, which has been previously reported(Malovannaya et al., Cell 145, 787-799, 2011).

Binding of DNMDP altered the composition of interacting proteins thatwere co-immunoprecipitated with PDE3A. Proteins that were specificallyenriched in PDE3A immunoprecipitates after treatment with DNMDP includedSirtuin 7 (SIRT7) and Schlafen 12 (SLFN12) (FIG. 13C and FIG. 14B).These proteins specifically interacted with PDE3A in the presence ofDNMDP, and were not observed in the trequinsin treated control, whereasa known PDE3B interactor, abhydrolase domain-containing protein 15(ABHD15, Chavez et al., Biochem. Biophys. Res. Commun. 342, 1218-1222,2006), was enriched in the immunoprecipitate from trequinsin-treatedcells (FIG. 13C and FIG. 14C). The interaction promoted by DNMDP betweenPDE3A and both SIRT7 and SLFN12 was validated with affinity reagents.Immunoprecipitation of endogenous PDE3A in HeLa cells treated withDNMDP, but not DMSO or trequinsin, enhanced complex formation ofectopically expressed V5-tagged SIRT7 and SLFN12 with PDE3A, asevidenced by coimmunoprecipitation (FIG. 19). FIG. 20 further shows thatDNMDP and (weakly) anagrelide, but not trequinsin, induced PDE3A andSFLN12 complex formation.

Similar to PDE3A, overexpression of SLFN12 appears to have a cytotoxiceffect in DNMDP sensitive cell lines, contributing to the difficulty ofdetecting SLFN12 in whole cell lysates.

The enhanced interaction of PDE3A with SIRT7 and SLFN12 indicated thepossibility that one or more of these interacting proteins mightcontribute to DNMDP sensitivity. SIRT7 mRNA expression was relativelyconstant among all cells tested, but the co-expression of SLFN12 andPDE3A mRNA showed a strong correlation with DNMDP sensitivity; almostall DNMDP-sensitive cell lines expressed high levels of SLFN12 (FIG.15A-15C). Importantly, almost half of sensitive cell lines expressinghigh levels of SLFN12 and PDE3A were found to be melanoma cell lines(FIG. 15B). SLFN12 expression alone was also one of the top genescorrelating with sensitivity to DNMDP, corroborating the hypothesis thatSLFN12 could be functionally involved in DNMDP-induced cytotoxicity(FIG. 16A). Moreover, when correcting for PDE3A expression, SLFN12expression was the top correlating gene with DNMDP sensitivity (FIG.16B). To assess whether SLFN12 is required for the cytotoxic phenotypeof DMNDP, we reduced SLFN12 mRNA expression by 60% by knockdown with twoshRNAs in HeLa cells (FIG. 15D). Similar to reduction in PDE3Aexpression, reduction of SLFN12 expression did not result incytotoxicity, and in fact decreased sensitivity to DNMDP (FIG. 15E).These results show that SLFN12, like PDE3A, is required for thecytotoxic phenotype of DMNDP. Characterization of normal expression ofSLFN12 and PDE3A by the GTEX consortium (Pierson, E. et al. PLoS Comput.Biol. 11, e1004220 (2015)) shows low expression of SLFN12 in normaltissues, while high co-expression of both PDE3A and SLFN12 is rarelyobserved (Table 3). This could suggest that on-target toxicity of DNMDPand related compounds may be potentially limited.

TABLE 3 RPKM values of SLFN12 and PDE3A expression in multiple healthytissue types SLFN12 PDE3A (RPKM) (RPKM) Mean SD Mean SD n Adipose -Subcutaneous 2.14 0.70 4.76 2.03 128 Adipose - Visceral 2.43 1.03 4.261.94 31 (Omentum) Adrenal Gland 3.01 0.83 0.34 0.21 52 Artery - Aorta2.10 0.71 16.15 5.12 82 Artery - Coronary 1.80 0.80 17.73 6.52 44Artery - Tibial 1.09 0.49 22.97 6.35 137 Bladder 1.38 0.57 1.33 0.40 11Brain - Amygdala 0.37 0.23 0.96 0.34 26 Brain - Anterior cingulate 0.280.16 0.77 0.45 22 cortex (BA24) Brain - Caudate (basal 0.40 0.23 1.270.37 36 ganglia) Brain - Cerebellar 0.11 0.07 2.73 1.49 29 HemisphereBrain - Cerebellum 0.19 0.10 2.40 0.98 31 Brain - Cortex 0.25 0.12 0.560.59 25 Brain - Frontal Cortex (BA9) 0.26 0.15 0.54 0.33 28 Brain -Hippocampus 0.39 0.31 0.82 0.38 28 Brain - Hypothalamus 0.46 0.29 0.930.48 30 Brain - Nucleus accumbens 0.28 0.16 1.11 0.41 32 (basal ganglia)Brain - Putamen (basal 0.29 0.18 0.91 0.33 24 ganglia) Brain - Spinalcord (cervical 0.50 0.32 0.65 0.55 19 c-1) Brain - Substantia nigra 0.620.50 0.82 0.47 27 Breast - Mammary Tissue 2.48 0.74 3.19 2.35 66 Cells -EBV-transformed 4.70 1.57 0.02 0.01 54 lymphocytes Cells - Transformed5.34 2.27 0.58 0.60 155 fibroblasts Colon - Sigmoid 1.58 0.50 10.27 3.4513 Colon - Transverse 0.99 0.47 11.24 4.32 45 Esophagus - 1.14 0.3116.87 5.53 22 Gastroesophageal Junction Esophagus - Mucosa 1.01 0.450.82 1.32 106 Esophagus - Muscularis 1.29 0.35 15.71 6.02 99 FallopianTube 2.32 0.86 3.80 1.86 6 Heart - Atrial Appendage 1.05 0.38 15.65 6.3138 Heart - Left Ventricle 0.81 0.38 26.55 13.43 95 Kidney - Cortex 1.211.07 1.40 0.84 8 Liver 0.29 0.16 0.49 0.28 34 Lung 2.83 1.12 2.78 1.48133 Minor Salivary Gland 1.75 0.61 0.62 0.44 5 Muscle - Skeletal 0.250.18 0.84 0.42 157 Nerve - Tibial 2.82 0.87 3.39 1.71 114 Ovary 1.920.57 2.17 1.13 35 Pancreas 0.52 0.27 2.65 0.86 65 Pituitary 0.47 0.231.04 0.47 22 Prostate 1.41 0.57 4.04 3.74 42 Skin - Not Sun Exposed 0.760.37 0.66 0.34 41 (Suprapubic) Skin - Sun Exposed (Lower 0.63 0.31 1.000.69 126 leg) Small Intestine - Terminal 1.61 0.72 7.34 4.83 17 IleumSpleen 3.46 0.92 1.18 0.46 34 Stomach 1.10 0.40 3.93 5.35 81 Testis 0.490.19 0.43 0.20 60 Thyroid 3.19 0.96 2.59 1.34 120 Uterus 1.99 0.56 3.291.55 32 Vagina 1.39 1.39 2.49 2.49 34 Whole Blood 1.40 1.10 0.06 0.05191

FIG. 21 shows that SLFN12 is lost in cells that have acquired resistanceto DNMDP. Cell lines initially sensitive to DNMDP were made resistant bypersistent exposure to DNMDP and subsequently analyzed by RNA-seq. Onegene was downregulated in both HeLa and H2122: SLFN 12. Accordingly, areduction in levels of SLFN 12 indicates that cells have becomeresistant to DNMDP and other PDE3A modulators.

FIG. 22 shows sensitization of a DNMDP-resistant cell line by expressionof SLFN12 or expression of SFLN12 and PDE3A. Expression of SLFN12 wassufficient to confer DNMDP sensitivity to A549 cells. Adding PDE3Aexpression led to further sensitization.

Leiomyosarcomas are malignant smooth muscle tumors. Patient tumorsamples from leiomyosarcomas were analyzed for PDE3A and SLFN12expression to characterize sensitivity of leiomyosarcomas (LMS) toDNMDP. Leiomyosarcomas are thought to be sensitive to DNMDP due toprevalence among high purity TCGA samples expressing elevated levels ofPDE3A and SLFN12 (FIG. 23, Table 4). P value for association ofbiomarker expression with leiomyosarcoma lineage: 0.0001.

TABLE 4 Leiomyosarcomas Characterization Marker Expression IndicatesMarker Expression DNMDP Indicates DNMDP sensitive not sensitive LMS 1731 Not LMS 38 1516

Differential scanning fluorimetry (DSF) was used to demonstrate bindingof DNMDP to purified PDE3A catalytic domain, PDE3A(677-1141). In thisexperiment, 5 μM hsPDE3A(640-1141) was incubated in the absence orpresence of 100 μM compounds, as indicated in Table 5. Binding buffer:20 mM Hepes pH 7.4, 100 μM TCEP, 1 mM MgCl₂, 150 mM NaCl.

TABLE 5 Binding of DNMDP to PDE3A(677-1141) T_(m) (° C.) ΔT_(m) (° C.)PDE3A₆₇₇₋₁₁₄₁ 52.4 ± 0.0 PDE3A₆₇₇₋₁₁₄₁ + DNMDP 58.4 ± 0.0 6.0PDE3A₆₇₇₋₁₁₄₁ + Anagrelide 56.6 ± 0.0 4.2 PDE3A₆₇₇₋₁₁₄₁ + Trequinsin66.2 ± 0.0 14.2

Using chemogenomics, a class of compounds was discovered, exemplified byDNMDP, that targeted a novel cancer dependency by small-moleculemodulation of PDE3A. These compounds bound PDE3A in a mutually exclusivemanner with non-cytotoxic PDE3 inhibitors and exerted a neomorphic orhypermorphic effect on the function of PDE3A, leading to a change in itsprotein-protein interactions. One unique protein-interaction partner,SLFN12, was highly expressed in DNMDP-sensitive cell lines, indicating afunctional role in the pathway through which the cytotoxic signal wasrelayed. As a result, DNMDP was both selective and potent across a largepanel of cancer cell lines.

Here, a novel cytotoxic compound was identified with great selectivityand low-nM potency against cancer cell lines across multiple lineages.Using gene-expression correlates for chemogenomics, PDE3A was identifiedas the putative target of this small molecule, DNMDP. Interestingly,loss of PDE3A expression resulted in resistance to DNMDP. Moreover,PDE3A immunoprecipitation followed by isobaric stable isotope tags forrelative abundance and quantitation by mass spectrometry (iTRAQ/MS)identified SLFN12 and SIRT7 as novel protein-protein interactionpartners of PDE3A upon DNMDP binding, possibly due to allostericmodulation of the function of PDE3A. Importantly, SLFN12 expression wasthe top correlating gene with DNMDP sensitivity when corrected for PDE3Aexpression. Single gene or multi-gene expression correlations have shownto help elucidate the mechanism of action and relevant signalingpathways of small molecules. A novel biochemical target for cancertreatment was identified that is unlikely to have been found by targetidentification approaches such as loss-of-function screens or genomicanalysis.

PDE3A belongs to the superfamily of phosphodiesterases and together withPDE3B forms the PDE3 family. The PDE3 family has dual substrate affinityand hydrolyses both cAMP and cGMP. Expression of PDE3A is highest in thecardiovascular system, platelets, kidney, and oocytes (Ahmad et al.,Horm Metab Res 44, 776-785, 2012). The clinical PDE3 inhibitorcilostazol has been developed to treat intermittent claudication, asPDE3A inhibition in platelets impairs activation and plateletcoagulation (Bedenis et al., Cochrane Database Syst Rev 10, CD003748,2014). Other PDE3 inhibitors, such as milrinone, amrinone, andlevosimendan, are indicated to treat congestive heart failure, where thecombination of vasodilation and elevated cardiac cAMP levels increasescardiac contractility (Movsesian et al., Curr Opin Pharmacol 11,707-713, 2011). None of these clinical inhibitors were able to replicatethe cytotoxic phenotype of DNMDP, indicating that cyclic nucleotidehydrolysis was not sufficient to induce cell death in DNMDP-sensitivecell lines.

Interestingly however, other PDE3 inhibitors such as zardaverine,anagrelide, and quazinone have been reported previously to have cellcytotoxic characteristics in a select number of cancer cell lines (Sunet al., PLoS ONE 9, e90627, 2014; Fryknäs et al., J Biomol Screen 11,457-468, 2006). In concordance with the present findings, other PDE3 andPDE4 inhibitors were found not to replicate the cytotoxic phenotype ofzardaverine where retinoblastoma protein retinoblastoma 1 (RB1)expression was reported to separate zardaverine sensitive cell linesfrom non-sensitive cell lines (Sun et al., PLoS ONE 9, e90627, 2014).This finding was in contrast to the present data where a correlationbetween cytotoxic activities of DNMDP and copy-number or mRNA expressionof RB1 was not identified. Another PDE3 inhibitor, anagrelide, uniquelyinhibited megakaryocyte differentiation, resulting in apoptosis. OtherPDE3 inhibitors tested did not have this activity (Wang et al., Br. J.Pharmacol. 146, 324-332, 2005; Espasandin, Y. et al., J. Thromb.Haemost. n/a-n/a, 2015, doi:10.1111/jth.12850). It was hypothesized thatthe reported effects of zardaverine on cell viability and anagrelide onmegakaryocyte differentiation are mediated through the same PDE3Amodulation as described in this study.

Multiple PDE3 inhibitors were competitive inhibitors and have been shownto occupy the catalytic binding site of cAMP and cGMP (Card et al.,Structure 12, 2233-2247, 2004; Zhan et al., Mol. Pharmacol. 62, 514-520,2002). In addition, zardaverine has been co-crystalized in a complexwith PDE4D, where it occupies the cAMP-binding site, and has beenmodeled to bind PDE3B in a similar manner (Lee et al., FEBS Lett. 530,53-58, 2002). Given the structural similarity of DNMDP to zardaverineand that DNMDP inhibited both PDE3A and PDE3B, it was hypothesized thatthe binding mode of DNMDP is very similar to that of zardaverine. Thisindicated that in addition to acting as a cAMP/cGMP-competitiveinhibitor, DNMDP allosterically induces a conformation that isresponsible for its cytotoxic phenotype. Allosteric modulation ofphosphodiesterases has been described previously for PDE4, where smallmolecules bound in the active site and simultaneously interacted withregulatory domains that came across the PDE4 active site. As a result,allosteric modulators stabilized a protein conformation that has beenshown to differentially bind different PDE4 partner proteins (Burgin etal., Nat Biotechnol 28, 63-70, 2010).

The study of proteins associated with PDE3A might illuminate both itsnormal function and the way in which PDE3A modulators such as DNMDP killcancer cells. PDE3A interacted with protein phosphatase 2 subunits,which are implicated in oncogenic viral transformation and are mutatedin human cancers (Nagao et al., Int. Symp. Princess Takamatsu CancerRes. Fund 20, 177-184, 1989; Imielinski et al., Cell 150, 1107-1120,2012; Lawrence et al., Nature 499, 214-218, 2013), indicating a role forPDE3A in cancer cell signaling. Even though these interactions were notinduced by DNMDP binding, the importance of the protein phosphatases incancer biology would warrant further research.

The enhanced interaction between PDE3A and SLFN12, facilitated by DNMDPbinding to PDE3A, and the correlation between sensitivity to DNMDP withSLFN12 expression strongly indicated that it is necessary to understandthe functional impact of the PDE3A-SLFN12 interaction. However, littleis known at this time about the functional role of SLFN12 in humanphysiology and cancer biology. SLFN12 is part of the schlafen genefamily that diverges largely between humans and rodents. The largedifference is due to rapid gene evolution and positive selection (Bustoset al., Gene 447, 1-11, 2009). Therefore, SLFN12 has no murineorthologue, preventing the study of SLFN12 in a well-understood modelorganism. The single publication on SLFN12 showed modulation of prostatecancer cell lines after ectopic expression of SLFN12 (Kovalenko et al.,J. Surg. Res. 190, 177-184, 2014). Additional studies into the functionof SLFN12 and its interaction with PDE3A could elucidate the mechanismof DNMDP cytotoxicity. Two observations indicated that DNMDP acted as aneomorph or hypermorph on PDE3A function: 1) DNMDP-sensitive cancer celllines did not depend on PDE3A expression for survival, but rather PDE3Aknock-down led to DNMDP resistance; and 2) DNMDP induced or enhancedprotein-protein interactions upon binding to PDE3A. Lenalidomide was anexample of a small molecule that acted as a neomorph or hypermorphrather than as an enzymatic inhibitor. Lenalidomide modulated a specificprotein-protein interaction between the cereblon ubiquitin ligase andIkaros transcription factors, which were then subsequently targeted fordegradation (Krönke et al., Science 343, 301-305, 2014; Lu et al.,Science 343, 305-309, 2014). By analogy, DNMDP might directly stabilizea PDE3A-SLFN12 interaction, or DNMDP could allosterically stabilize aPDE3 conformation that binds SLFN12. Either of these mechanisms couldresult in a neo- or hypermorphic phenotype. Further characterization ofthe neomorphic phenotype induced by DNMDP might facilitate synthesis ofsmall molecules that will not inhibit cyclic nucleotide hydrolysis byPDE3A. Toxicity profiles of such small molecules should differ from PDE3inhibitors prescribed for cardiovascular indications.

This study has uncovered a previously unknown role for PDE3A in cancermaintenance, in which its function can be modified by a subset of PDE3inhibitors, resulting in toxicity to a subset of cancer cell lines.These data indicated that DNMDP and its analogs had a hyper- orneomorphic effect on PDE3A, leading to cellular toxicity, which wascorroborated by cells becoming less sensitive to DNMDP with decreasinglevels of cellular PDE3A. These observations are comparable with otherreports of allosteric modulation of phosphodiesterases (Burgin et al.,Nat Biotechnol 28, 63-70, 2010), indicating that DNMDP and analogues mayhave similar effects on PDE3A. The exact mechanism of cell-selectivecytotoxicity remains unknown for now; however, further studies into thenovel interactions with SLFN12, and perhaps SIRT7, might be informative.

In summary, the study herein used differential cytotoxicity screening todiscover a cancer cell cytotoxic small molecule, DNMDP. Profiling ofDNMDP in 766 genomically-characterized cancer cell lines revealedstereospecific nanomolar efficacy in about 3% of cell lines tested. Asearch for genomic features that indicated sensitivity revealed thatelevated PDE3A expression strongly correlated with DNMDP response. DNMDPinhibited PDE3A and PDE3B, with little or no activity towards otherPDEs. However, unexpectedly, most other PDE3A inhibitors tested did notphenocopy DNMDP, including the potent and selective PDE3A inhibitor,trequinsin. Co-treatment of DNMDP-sensitive cells with trequinsincompeted away the cancer cell cytotoxic activity of DNMDP, and knockoutof PDE3A rescued the otherwise sensitive cells from DNMDP-inducedcytotoxicity, leading us to hypothesize that PDE3A is required forcancer cell killing by DNMDP, which induces a neomorphic alteration ofPDE3A. Mass spectrometric analysis of PDE3A immunoprecipitates alone orin the presence of DNMDP or trequinsin revealed differential binding ofSLFN12 and SIRT7 only in the presence, of DNMDP. Similar to PDE3A,SLFN12 expression levels were elevated in DNMDP-sensitive cell lines,and knock down of SLFN12 with shRNA decreased sensitivity of cells toDNMDP, indicating that DNMDP-induced complex formation of PDE3A withSLFN12 is critical to the cancer cell cytotoxic phenotype. Resultsherein therefore implicate PDE3A modulators as candidate cancertherapeutic agents and demonstrate the power of chemogenomics in smallmolecule discovery.

The experiments above were performed with the following methods andmaterials.

Compound Library Screening in NCI-H1734 and A549 Cell Lines

1500 NCI-H1734 or 1000 A549 cells were plated in a 384-well plate in 40μl of RPMI supplemented with 10% Fetal Bovine Serum and 1% Pen/Strep. 24hours after plating, a compound-library of 1924 small molecules wasadded at a concentration of 10 μM. Staurosporine was used a positivecontrol for cytotoxicity at a concentration of 10 μM, and DMSO was useda negative control at a concentration of 1%. All compounds wereincubated for 48 hours with indicated small molecules. After 48 hours,384-well plates were removed from the incubator and allowed to cool toroom temperature for 20 minutes. Cell viability was assessed by adding40 μl of a 25% CELLTITERGLO® (Promega) in PBS with a THERMO COMBI™ ormultichannel-pipette and incubated for 10 minutes. The luminescencesignal was read using a Perkin-Elmer EnVision. Viability percentage wascalculated by normalizing to DMSO controls.

Compound Sensitivity Testing in Cell Lines

1000 HeLa (DMEM), 1000 A549 (RPMI), 500 MCF-7 (DMEM), 4000 PC3 (F12-K),1000 NCI-H2122 (RPMI) or 1500 NCI-H1563 (RPMI) cells were plated in a384-well plate in 40 μl of corresponding growth media supplemented with10% Fetal Bovine Serum. 24 hours after plating, indicated compounds wereadded at indicated concentrations and incubated for 48 hours. Cellviability was assessed as described in Compound library screening inNCI-H1734 and A549 cell lines.

Caspase Activity in HeLa Cells

1000 HeLa cells were plated in 384-well plate in 40 μl of correspondinggrowth media supplemented with 10% Fetal Bovine Serum. 24 hours afterplating, indicated compounds were added at indicated concentrations andincubated for 48 hours. Caspase-Glo from Promega was added according tothe manufacturers recommendations and luminescence was determined asdescribed in Compound library screening in NCI-H1734 and A549 celllines.

Large-Scale Cell-Line Viability Measurements

The sensitivity of 777 cancer cell lines (CCLs) was measured drawn from23 different lineages to DNMDP. Each cell line was plated in itspreferred media in white opaque 1536-plates at a density of 500cells/well. After incubating overnight, DNMDP was added by acoustictransfer at 16 concentrations ranging from 66.4 μM-2 nM in 2-fold stepsin duplicate (Labcyte Echo 555, Labcyte Inc., Sunnyvale, Calif.). After72 hours treatment, cellular ATP levels were measured as a surrogate forviability (CELLTITERGLO®, Promega Corporation, Madison, Wis.) accordingto manufacturer's protocols using a ViewLux Microplate Imager(PerkinElmer, Waltham, Mass.) and normalized to background (media-only)and vehicle (DMSO)-treated control wells.

Concentration response curves were fit using nonlinear fits to 2- or3-parameter sigmoid functions through all 16 concentrations with thelow-concentration asymptote set to the DMSO-normalized value, and anoptimal 8-point dose curve spanning the range of compound-sensitivitywas identified. The area under the 8-point dose curve (AUC) was computedby numeric integration as a metric for sensitivity for further analysis.Similar sensitivity measurements have been obtained for a collection of480 other compounds, enabling analyses that identify cell linesresponding uniquely to DNMDP (see Broad Institute Cancer TherapeuticsResponse Portal, a dataset to identify comprehensively relationshipsbetween genetic and lineage features of human cancer cell lines andsmall-molecule sensitivities for complete list of compounds).

Correlation of Sensitivity Measurements with Basal Gene Expression

Gene-centric robust multichip average (RMA)-normalized basal mRNA geneexpression data measured on the Affymetrix GeneChip Human Genome U133Plus 2.0 Array were downloaded from the Cancer Cell Line Encyclopedia(CCLE, a detailed genetic characterization of a large panel of humancancer cell lines; Barretina et al., Nature 483, 603-607, 2012). Pearsoncorrelation coefficients were calculated between gene expression (18,988transcripts) and areas under the curve (AUCs) across 760 overlappingCCLs. For comparisons across small molecules exposed to differingnumbers of CCLs, correlation coefficients were transformed usingFisher's transformation.

Chemistry Experimental Methods

General Details

All reactions were carried out under nitrogen (N2) atmosphere. Allreagents and solvents were purchased from commercial vendors and used asreceived. Nuclear magnetic resonance (NMR) spectra were recorded on aBruker (300 or 400 MHz ¹H, 75 or 101 MHz ¹³C) spectrometer. Proton andcarbon chemical shifts are reported in ppm (δ) referenced to the NMRsolvent. Data are reported as follows: chemical shifts, multiplicity(br=broad, s=singlet, d=doublet, t=triplet, q=quartet, m=multiplet;coupling constant(s) in Hz). Flash chromatography was performed using40-60 μm Silica Gel (60 Å mesh) on a Teledyne Isco Combiflash Rf. TandemLiquid Chromatography/Mass Spectrometry (LC/MS) was performed on aWaters 2795 separations module and 3100 mass detector with a WatersSymmetry C18 column (3.5 μm, 4.6×100 mm) with a gradient of 0-100% CH3CNin water over 2.5 min with constant 0.1% formic acid. Analytical thinlayer chromatography (TLC) was performed on EM Reagent 0.25 mm silicagel 60-F plates. Elemental analysis was performed by Robertson MicrolitLaboratories, Ledgewood N.J.

Synthesis of (R)-DNMDP

In 5 mL of acetic anhydride, 2.00 g (9.84 mmol) of(R)-6-(4-aminophenyl)-5-methyl-4,5-dihydropyridazin-3(2H)-one (A,Toronto Research Chemicals) was stirred 1 hour before addition of 30 mLwater, filtration, rinsing the solids with water and drying to yield2.20 g of product B (91%). ¹H NMR (300 MHz, DMSO-d₆) δ 10.92 (s, 1H),10.13 (s, 1H), 7.74 (d, J=8.9, 2H), 7.65 (d, J=8.8, 2H), 3.41-3.33 (m,1H), 2.68 (dd, J=6.8, 16.8, 1H), 2.23 (d, J=16.7, 1H), 2.08 (s, 3H),1.07 (d, J=7.3, 3H). ¹³C NMR (75 MHz, DMSO-d₆) δ 168.50, 166.27, 152.25,140.27, 129.24, 126.24, 118.70, 33.47, 26.91, 24.02, 15.87. HPLC: R_(t)0.72 min, purity >95%. MS: 246 (M+1).

To 3.09 g of B (15.3 mmol) dissolved in 30 mL of sulfuric acid andcooled in an ice bath was added 0.72 mL of 90% nitric acid (15 mmol) in8 mL sulfuric acid via an addition funnel over 10 minutes. Afterstirring 1 hour the mixture was poured onto ice. The yellow solid wasfiltered off and the water was rinsed several times with EtOAc beforedrying and combining with the yellow solid. Chromatography with 40-60%EtOAc in hexane yielded 1.12 g (25%) of product as a yellow solid whichwas recrystallized from EtOAc. ¹H NMR (300 MHz, DMSO-d₆) δ 11.13 (s,1H), 10.41 (s, 1H), 8.25 (d, J=1.8, 1H), 8.07 (dd, J=1.8, 8.6, 1H), 7.71(d, J=8.6, 1H), 3.55-3.40 (m, 1H), 2.74 (dd, J=6.9, 16.8, 1H), 2.27 (d,J=16.8, 1H), 2.09 (s, 3H), 1.08 (d, J=7.2, 3H). ¹³C NMR (75 MHz,DMSO-d6) δ 168.57, 166.31, 150.37, 142.19, 131.69, 131.32, 130.60,125.07, 121.70, 33.30, 26.81, 23.44, 15.64. TLC: Rf 0.25 (1:1EtOAc:hexane). HPLC: R_(t) 0.87 min, purity >95%. MS: 291 (M+1). HRMSExact Mass (M+1): 291.1088. Found: 291.1091.

To 58 mg of C (0.20 mmol) dissolved in 10 mL of MeOH was added asolution of 48 mg NaOH (1.2 mmol) in 0.5 mL water. After 1 hour thereaction was concentrated, water was added and rinsed with EtOAc, theEtOAc was dried and concentrated to give 48 mg (93%) of product D. ¹HNMR (300 MHz, DMSO-d6) δ 10.92 (s, 1H), 8.28 (d, J=2.0, 1H), 7.87 (dd,J=2.1, 9.0, 1H), 7.76 (s, 2H), 7.06 (d, J=9.0, 1H), 3.33 (s, 1H), 2.67(dd, J=6.8, 16.8, 1H), 2.22 (d, J=16.6, 1H), 1.06 (d, J=7.3, 3H). ¹³CNMR (75 MHz, DMSO-d₆) δ 166.25, 151.12, 146.69, 132.72, 129.80, 122.57,122.19, 119.80, 33.43, 26.70, 15.77. MS: 249 (M+1).

To 35 mg of amine D (0.14 mmol) dissolved in 0.5 mL Dimethylformamide(DMF) was added 70 mg of acetaldehyde (1.6 mmol) and 170 mg ofNaBH(OAc)₃ (0.80 mmol) and 10 μL, (0.2 mmol) of HOAc. After stirring 3hours, water and EtOAc were added, the EtOAc separated, dried,concentrated and chromatographed with 30-50% EtOAc in hexane to isolate3 mg of the (R)-DNMDP (7%). The synthesized material was identical topurchased racemic material by TLC, HPLC and ¹H NMR. ¹H NMR (300 MHz,CDCl₃) δ 8.58 (s, 1H), 8.04 (d, J=2.3, 1H), 7.84 (dd, J=2.3, 9.0, 1H),7.11 (d, J=9.0, 1H), 3.30-3.36 (m, 1H), 3.26 (q, J=7.1, 4H), 2.71 (dd,J=6.8, 16.9, 1H), 2.48 (d, J=17.0, 1H), 1.25 (d, J=7.4, 3H), 1.16 (t,J=7.1, 6H). TLC: Rf 0.25 (1:1 EtOAc:hexane). HPLC: R_(t) 1.27 min,purity >95%. MS: 305 (M+1). Exact Mass (M+1): 305.1608 Found: 305.1616.¹³C NMR (75 MHz, CDCl₃, purchased material) δ 166.28, 152.02, 145.24,141.21, 129.77, 124.94, 123.94, 121.00, 46.10, 33.80, 27.81, 16.24,12.56.

The optical purity of (R)-DNMDP was determined using chiral SCFchromatography and comparison to commercially available racemicmaterial: Column: ChiralPak AS-H, 250×4.6 mm, 5 μm, Mobile PhaseModifier: 100% Methanol, Gradient: 5 to 50% Methanol over 10 minutes,Flow Rate: 4 mL/min, Back Pressure: 100 bar, Column Temperature: 40 C.UV detection was from 200-400 nm. Retention times of separated isomers:5.36, 6.64 minutes; retention time of (R)-DNMDP, 6.60 minutes, 1:19ratio of enantiomers detected.

2. To 200 mg (0.98 mmol) of A dissolved in 5 mL of MeOH was added 87 mgof acetaldehyde (2.0 mmol), 113 uL of HOAc (2.0 mmol) and 124 mg (2.0mmol) of NaBH₃CN and the reaction was stirred overnight at roomtemperature. The next day the same quantity of reagents were added andthe reaction stirred another 24 hours. The mixture was concentrated andpartitioned between CH₂Cl₂ and water, the CH₂Cl₂ was separated, dried,and concentrated before chromatography with 20-40% EtOAc in hexaneisolated 210 mg of product as a white solid (82%). ¹H NMR (300 MHz,CDCl₃) δ 8.95 (s, 1H), 7.64 (d, J=8.7, 2H), 6.66 (d, J=8.7, 2H), 3.37(dd, J=9.6, 16.4, 5H), 2.67 (dd, J=6.5, 16.8, 1H), 2.43 (d, J=16.8, 1H),1.41-1.02 (m, 10H). ¹³C NMR (75 MHz, CDCl₃) δ 166.82, 154.55, 148.79,127.32, 120.81, 111.08, 44.32, 33.92, 27.74, 16.37, 12.50. TLC: Rf 0.25(1:1 EtOAc:hexane). HPLC: R_(t) 1.05 min, purity >95%. MS: 260 (M+1).HRMS Exact Mass (M+1): 260.1757. Found: 260.1764

3. To 200 mg (0.984 mmol) of A dissolved in 1 mL of Dimethylformamide(DMF) was added 250 μL (2.00 mmol) of bis (2-bromoethyl) ether and 400mg of K2CO3 and the mixture was stirred overnight at 60° C. The next dayanother 250 μL of bis (2-bromoethyl) ether and 170 mg of K2CO3 wereadded. After 3 hours, EtOAc and water were added, the water was rinsedwith EtOAc, the combined EtOAc washes were dried and concentrated.Chromatography with 0-4% MeOH in CH2Cl2 yielded 125 mg of product (46%).¹H NMR (300 MHz, CDCl₃) δ 8.61 (s, 1H), 7.68 (d, J=8.8, 2H), 6.92 (d,J=8.8, 2H), 3.99-3.76 (m, 4H), 3.44-3.31 (m, 1H), 3.29-3.22 (m, 4H),2.70 (dd, J=6.7, 16.8, 1H), 2.46 (d, J=16.7, 1H), 1.24 (d, J=7.3, 3H).¹³C NMR (75 MHz, CDCl₃) δ 166.64, 154.05, 152.18, 127.10, 125.33,114.73, 66.69, 48.33, 33.93, 27.94, 16.36. TLC: R_(f) 0.1 (1:50MeOH:CH₂Cl₂). HPLC: R_(t) 1.05 min, purity >95%. MS: 274 (M+1). HRMS:calcd. 274.1556 (M+1); found 274.1552. Anal. Calcd. for C₁₅H₁₉N₃O₂: C,65.91; H, 7.01; N, 15.37; Found. 65.81, H, 6.66, N, 15.26.

DNMDP-2L.

To 130 mg of A (0.64 mmol) dissolved in 0.4 mL of Dimethylformamide(DMF) was added 100 mg of tert-butyl2-(2-(2-bromoethoxy)ethoxy)-ethylcarbamate (Toronto Research Chemical,0.32 mmol) and 90 mg of K₂CO³ (64 mmol) and the mixture was stirred at60° C. overnight. After cooling, water was added and rinsed severaltimes with EtOAc. The combined EtOAc layers were dried, concentrated,and chromatographed with 50-70% EtOAc to yield 81 mg of product (58%).NMR (300 MHz, CDCl₃) δ 9.06 (s, 1H), 7.59 (d, J=8.8 Hz, 2H), 6.62 (d,J=8.8 Hz, 2H), 5.15 (s, 1H), 4.53 (s, 1H), 3.72 (t, J=5.2 Hz, 2H), 3.65(s, 4H), 3.55 (t, J=5.2 Hz, 2H), 3.32 (m, 5H), 2.67 (dd, J=16.8, 6.7 Hz,1H), 2.42 (d, J=16.4 Hz, 1H), 1.44 (s, 9H), 1.22 (d, J=7.4 Hz, 3H). ¹³CNMR (75 MHz, CDCl₃) δ 166.83, 155.99, 154.45, 149.64, 127.33, 123.24,112.58, 79.28, 70.30, 70.26, 70.22, 69.45, 43.14, 40.39, 33.96, 28.43,27.89, 16.40; HPLC: R_(t)2.50 min (7.5 min run), purity >95%. MS: 435(M+1). This product (0.19 mmol) was dissolved in 1 mL MeOH and to thesolution was added acetaldehyde (50 uL, 0.89 mmol), 10 uL HOAc (0.2mmol) and 12 mg NaBH₃CN (0.19 mmol). After 1 hour, NaHCO₃(aq) and CH₂Cl₂were added, the CH₂Cl₂ was separated and the water washed twice withCH₂Cl₂. The combined CH₂Cl₂ was dried, concentrated, and chromatographywith 60-70% EtOAc in hexane yielded 71 mg of product as a clear oil(82%). ¹H NMR (400 MHz, CDCl₃) δ 8.91 (s, 1H), 7.63 (d, J=8.9 Hz, 2H),6.69 (d, J=8.9 Hz, 2H), 5.07 (s, 1H), 3.65 (t, J=6.0 Hz, 2H), 3.61 (s,4H), 3.55 (dt, J=9.9, 5.5 Hz, 4H), 3.46 (q, J=7.0 Hz, 2H), 3.38-3.22 (m,3H), 2.67 (dd, J=16.8, 6.7 Hz, 1H), 2.43 (d, J=16.7 Hz, 1H), 1.45 (s,10H), 1.23 (d, J=7.3 Hz, 3H), 1.18 (t, J=7.0 Hz, 3H). ¹³C NMR (101 MHz,CDCl₃) δ 166.84, 155.96, 154.46, 148.89, 127.35, 121.38, 111.28, 79.22,70.68, 70.27, 70.24, 68.74, 49.95, 45.49, 40.32, 33.97, 28.43, 27.80,16.43, 12.14. R_(t) 2.99 min (7.5 min run), purity >95%. MS: 463 (M+1).

Attachment to Resin

To a solution of 18 mg of DNMDP-2L (0.04 mmol) in 0.8 mL of CH₂Cl₂ wasadded 0.2 mL of trifluoroacetic acid (TFA) and the solution was stirred2 h before concentration and dissolution in 0.5 mL DMSO. To this wasadded 10 uL of Et₃N (0.07 mmol) and 12 mg of N,N′-disuccinimidylcarbonate (DSC) (0.05 mmol) and the solution was stirred overnight. LCanalysis indicated the reaction was not complete, another 25 mg ofN,N′-disuccinimidyl carbonate (0.1 mmol) was added. LC analysis after 2hours showed ca. 5:1 ratio of DSC product:amine. A 1 mL sample ofAffi-Gel 102 resin was rinsed five times with DMSO with a centrifuge,then suspended in 0.5 mL DMSO. To the resin was added 30 uL of the DSCproduct solution and 25 uL Et3N and the mixture was swirled. After 2days, LC analysis of the DMSO solution showed complete disappearance ofthe DCS adduct; the underivatized amine was still present. The DMSO wasremoved by centrifuge and decanted and the resin was rinsed severaltimes with DMSO and stored in PBS buffer.

Bioactives Screen to Rescue DNMDP Induced Cytotoxicity

1000 HeLa cells were plated in a 384-well plate in 40 μl of DMEMsupplemented with 10% Fetal Bovine Serum and 1% Pen/Strep. 24 hoursafter plating, a compound-library of 1600 bioactive molecules(Pharmacon) was added at a concentration of 20 μM. In parallel tobioactive compound incubation, DNMDP was added to a final concentrationof 30 nM and incubated for 48 hours. Cell viability was assessed asdescribed in Compound library screening in NCI-H1734 and A549 celllines.

Linker-Affinity Purification of Molecular Target of DNMDP andImmunoblotting

HeLa cells were washed with ice-cold PBS before lysed with NP-40 lysisbuffer (150 mM NaCl, 10% glycerol, 50 mM Tris-Cl pH 8.0, 50 mM MgCl₂, 1%NP-40) supplemented with EDTA-free protease inhibitors (Roche) andphosphatase inhibitor mixtures I and II (Calbiochem). Cell lysates wereincubated on ice for at least 2 minutes and subsequently centrifuged for10 minutes at 4° C. at 15,700×g after which the supernatant wasquantified using BCA protein assay kit (Pierce). 200 μg total HeLa celllysate was incubated with 3 μl Affi-Gel 102 resin (BioRad) coupled toaffinity linker DNMDP-2L in a total volume of 400 μl for four hours.Prior to incubation, indicated compounds were added to affinitypurifications at a final concentration of 10 μM. Samples were washedthree times with lysis buffer containing corresponding compoundconcentrations of 10 μM. Proteins bound to Affi-Gel 102 resin werereduced, denatured, and separated using Tris-Glycine gels (Novex) andtransferred to nitrocellulose membranes using the iBlot transfer system(Novex). Membranes were incubated overnight at 4° C. with primaryantibodies against PDE3A (1:1000, Bethyl). Incubation with secondaryantibodies (1:20,000, LI-COR Biosciences) for two hours at roomtemperature and subsequent detection (Odyssey Imaging System, LI-CORBiosciences) were performed according to manufacturer's recommendations.

PARP-Cleavage Immunoblotting

HeLa cells were treated with indicated concentration of DNMDP andstaurosporine for 36 hours. HeLa cells were lysed and processed asdescribed in Linker-affinity purification of molecular target of DNMDPand immunoblotting. Membranes were incubated with an antibody againstPARP (1:1000, Cell Signaling #9532) and actin and subsequently imaged asdescribed in Linker-affinity purification of molecular target of DNMDPand immunoblotting.

Targeting PDE3A Locus Using CRISPR

CRISPR target sites were identified using the MIT CRISPR Design Tool(online MIT CRISPR design portal). For cloning of sgRNAs, forward andreverse oligos were annealed, phosphorylated and ligated intoBsmBI-digested pXPR_BRD001. Oligo sequences are as follows:

sgRNA Forward oligo Reverse oligo PDE3A_sg1 CACCGTTTTCACTGAGCGAAGTGAAAACTCACTTCGCTCAGTGAAAAC (SEQ ID NO.: 7) (SEQ ID NO.: 8) PDE3A_sg2CACCGAGACAAGCTTGCTATTCCAA AAACTTGGAATAGCAAGCTTGTCTC (SEQ ID NO.: 9)(SEQ ID NO.: 10) PDE3A_sg3 CACCGGCACTCTGAGTGTAAGTTAAAACTAACTTACACTCAGAGTGCC (SEQ ID NO.: 11) (SEQ ID NO.: 12)To produce lentivirus, 293T cells were co-transfected with pXPR_BRD001,psPAX2 and pMD2.G using calcium phosphate. Infected HeLa cells wereselected with 2 ug/ml of puromycin.Reduction of PDE3A Expression Using siRNA

HeLa cells were plated in 96-well plates and transfected after 24 hourswith PDE3A and Non-Targeting siRNA smartpools (On Target Plus, ThermoScientific) according to the manufacturers recommendations. HeLa celllysate was obtained 24 hours and 72 hours after transfection andimmunoblotted for PDE3A and Actin (1:20,000, Cell Signaling) asdescribed in Linker-affinity purification of molecular target of DNMDPand immunoblotting. HeLa cells were treated for 48 hours with indicatedconcentrations of Compound 3. Cell viability was assessed as describedin Compound library screening in NCI-H1734 and A549 cell lines.

Measuring Cellular cAMP Concentrations in HeLa Cells

5000 HeLa cells were plated in 96-well plates. 24 hours after plating,HeLa cells were incubated for one hour with indicated compounds atindicated concentrations. cAMP levels were determined with the CAMP-GLO™assay (Promega) according to the manufacturers recommendations. Cellularconcentrations of cAMP were determined by normalizing to a standardcurve generated according to the manufacturers recommendations.

Extended Proteomics Methods for PDE3A-Protein Interaction Studies

Immunoprecipitation of PDE3A in HeLa Cells

HeLa cells were treated for four hours prior to lysis with 10 μM ofindicated compounds: DMSO, DNMDP and trequinsin. HeLa cells were lysedwith ModRipa lysis buffer (1% NP-40:50 mM Tris-HCl, pH 7.8, 150 mM NaCl,0.1% sodium deoxycholate, 1 mM EDTA) supplemented with protease andphosphatase inhibitors as described in Linker-affinity purification ofmolecular target of DNMDP and immunoblotting, and indicated compounds asdescribed above to a final concentration of 10 μM. 13 mg of HeLa totalcell lysate was incubated with 0.5% PDE3A antibody (Bethyl) andincubated overnight. Blocking peptide (Bethyl) against the PDE3Aantibody was added simultaneously with the PDE3A antibody in thecorresponding condition. Total cell lysate and antibody mixture was thenincubated with 10 μl Protein A Plus Agarose (Fisher Scientific) for 30minutes at 4° C. Protein A Plus Agarose was then washed two times withlysis buffer containing indicated compounds at a concentration of 10 μM.Finally, Protein A Plus Agarose was washed once with lysis buffercontaining no NP-40 and indicated compounds at a concentration of 10 μM.

On-Bead Digest

The beads from immunopurification were washed once with IP lysis buffer,then three times with PBS, the three different lysates of each replicatewere resuspended in 90 uL digestion buffer (2M Urea, 50 mM Tris HCl), 2ug of sequencing grade trypsin added, 1 hour shaking at 700 rpm. Thesupernatant was removed and placed in a fresh tube. The beads were thenwashed twice with 50 uL digestion buffer and combined with thesupernatant. The combined supernatants were reduced (2 uL 500 mM DTT, 30minutes, room temperature), alkylated (4 uL 500 mM IAA, 45 minutes,dark) and a longer overnight digestion performed: 2 ug (4 uL) trypsin,shake overnight. The samples were then quenched with 20 uL 10% folicacid (FA) and desalted on 10 mg SEP-PAK® columns.

iTRAQ Labeling of Peptides and Strong Cation Exchange (SCX)Fractionation

Desalted peptides were labeled with isobaric tags for relative andabsolute quantification (iTRAQ)-reagents according to the manufacturer'sinstructions (AB Sciex, Foster City, Calif.). Peptides were dissolved in30 μl of 0.5 M TEAB pH 8.5 solution and labeling reagent was added in 70ul of ethanol. After 1 hour incubation the reaction was stopped with 50mM Tris/HCl pH 7.5. Differentially labeled peptides were mixed andsubsequently desalted on 10 mg SEP-PAK® columns.

iTRAQ labeling 114 115 116 117 Rep1 Blocking peptide No addition DNMDPtrequinsin Rep2 Blocking peptide No addition DNMDP trequinsinSCX fractionation of the differentially labelled and combined peptideswas done as described in Rappsilber et al. (Rappsilber et al., NatProtoc 2, 1896-1906, 2007), with 6 pH steps (buffers-all contain 25%acetonitrile) as below:

1: ammonium acetate 50 mM pH 4.5,

2: ammonium acetate 50 mM pH 5.5,

3: ammonium acetate 50 mM pH 6.5,

4: ammonium bicarbonate 50 mM pH 8,

5: ammonium hydroxide 0.1% pH 9,

6: ammonium hydroxide 0.1% pH 11.

Empore SCX disk used to make stop-and-go-extraction-tips (StageTips) asdescribed in the paper.

MS Analysis

Reconstituted peptides were separated on an online nanoflow EASY-NLC™1000 UHPLC system (Thermo Fisher Scientific) and analyzed on a benchtopOrbitrap Q EXACTIVE™ mass spectrometer (Thermo Fisher Scientific). Thepeptide samples were injected onto a capillary column (PICOFRIT® with 10μm tip opening/75 μm diameter, New Objective, PF360-75-10-N-5) packedin-house with 20 cm C18 silica material (1.9 μm REPROSIL-PUR® C18-AQmedium, Dr. Maisch GmbH, r119.aq). The UHPLC setup was connected with acustom-fit microadapting tee (360 μm, IDEX Health & Science, UH-753),and capillary columns were heated to 50° C. in column heater sleeves(Phoenix-ST) to reduce backpressure during UHPLC separation. Injectedpeptides were separated at a flow rate of 200 nL/min with a linear 80min gradient from 100% solvent A (3% acetonitrile, 0.1% formic acid) to30% solvent B (90% acetonitrile, 0.1% formic acid), followed by a linear6 min gradient from 30% solvent B to 90% solvent B. Each sample was runfor 120 minutes, including sample loading and column equilibrationtimes. The Q EXACTIVE™ instrument was operated in the data-dependentmode acquiring high-energy collisional dissociation (HCD) MS/MS scans(R=17,500) after each MS1 scan (R=70,000) on the 12 top most abundantions using an MS1 ion target of 3×106 ions and an MS2 target of 5×104ions. The maximum ion time utilized for the MS/MS scans was 120 ms; theHCD-normalized collision energy was set to 27; the dynamic exclusiontime was set to 20 s, and the peptide match and isotope exclusionfunctions were enabled.

Quantification and Identification of Peptides and Proteins

All mass spectra were processed using the Spectrum Mill software packagev4.1 beta (Agilent Technologies) which includes modules developed byApplicants for isobaric tags for relative and absolute quantification(iTRAQ)-based quantification. Precursor ion quantification was doneusing extracted ion chromatograms (XIC's) for each precursor ion. Thepeak area for the XIC of each precursor ion subjected to MS/MS wascalculated automatically by the Spectrum Mill software in theintervening high-resolution MS1 scans of the liquid chromatography(LC)-MS/MS runs using narrow windows around each individual member ofthe isotope cluster. Peak widths in both the time and m/z domains weredynamically determined based on MS scan resolution, precursor charge andm/z, subject to quality metrics on the relative distribution of thepeaks in the isotope cluster vs theoretical. Similar MS/MS spectraacquired on the same precursor m/z in the same dissociation mode within+/−60 seconds were merged. MS/MS spectra with precursor charge >7 andpoor quality MS/MS spectra, which failed the quality filter by nothaving a sequence tag length >1 (i.e., minimum of 3 masses separated bythe in-chain mass of an amino acid) were excluded from searching.

For peptide identification MS/MS spectra were searched against humanUniversal Protein Resource (Uniprot) database to which a set of commonlaboratory contaminant proteins was appended. Search parametersincluded: ESI-Q EXACTIVE™-HCD scoring parameters, trypsin enzymespecificity with a maximum of two missed cleavages, 40% minimum matchedpeak intensity, +/−20 ppm precursor mass tolerance, +/−20 ppm productmass tolerance, and carbamidomethylation of cysteines and iTRAQ labelingof lysines and peptide n-termini as fixed modifications. Allowedvariable modifications were oxidation of methionine, acetylation,Pyroglutamic acid (N-termQ), Deamidated (N), Pyro Carbamidomethyl Cys(N-termC), with a precursor MH+ shift range of −18 to 64 Da. Identitiesinterpreted for individual spectra were automatically designated asvalid by optimizing score and delta rank1-rank2 score thresholdsseparately for each precursor charge state in each liquid chromatography(LC)-MS/MS while allowing a maximum target-decoy-based false-discoveryrate (FDR) of 1.0% at the spectrum level.

In calculating scores at the protein level and reporting the identifiedproteins, redundancy is addressed in the following manner: the proteinscore is the sum of the scores of distinct peptides. A distinct peptideis the single highest scoring instance of a peptide detected through anMS/MS spectrum. MS/MS spectra for a particular peptide may have beenrecorded multiple times, (i.e. as different precursor charge states,isolated from adjacent SCX fractions, modified by oxidation of Met) butare still counted as a single distinct peptide. When a peptidesequence >8 residues long is contained in multiple protein entries inthe sequence database, the proteins are grouped together and the highestscoring one and its accession number are reported. In some cases whenthe protein sequences are grouped in this manner there are distinctpeptides which uniquely represent a lower scoring member of the group(isoforms or family members). Each of these instances spawns a subgroupand multiple subgroups are reported and counted towards the total numberof proteins. iTRAQ ratios were obtained from the protein-comparisonsexport table in Spectrum Mill. To obtain iTRAQ protein ratios the medianwas calculated over all distinct peptides assigned to a protein subgroupin each replicate. To assign interacting proteins the Limma package inthe R environment was used to calculate moderated t-test p, as describedpreviously and added Blandt-Altman testing to filter out proteins forwhich the CI for reproducibility was below 95% (Udeshi et al., Mol CellProteomics 11, 148-159, 2012).

Validation of DNMDP-Induced PDE3A Protein Interactions UsingImmunoprecipitation and Immunoblotting

HeLa cells were transfected with ORF overexpression constructsexpressing V5-tagged SIRT7, V5-tagged SLFN12, or V5-tagged GFP. ORFexpression constructs were obtained from the TRC (clone IDs:TRCN0000468231, TRCN0000476272, ccsbBroad304_99997). At 72 hours posttransfection, cells were treated with 10 μM DNMDP or trequinsin for 4hours followed by lysis using the ModRipa lysis buffer andimmunoprecipitation of PDE3A. For each condition, 2 mg total proteinlysate was incubated with 1 μg of anti-PDE3A antibody at 4° C.overnight, after which 7.5 μl each of Protein A- and Protein G-Dynabeads(Life Technologies 10001D and 10003D) were added and incubated foranother 1 hour. Beads were washed and bound proteins were eluted with 30μl of LDS PAGE gel loading buffer. Input (˜60 μg total protein lysate)and IP products were resolved on 4-12% Tris-Glycine PAGE gels andimmunoblotted with an anti-V5 antibody (Life Technologies R96205,1:5000), the Bethyl anti-PDE3A antibody (1:1000), and secondaryantibodies from LiCOR Biosciences (Cat.#926-32210 and 926068021, each at1:10,000). Blots were washed and imaged using a LiCOR Odyssey infraredimager.

Knockdown of SLFN12 Expression Using shRNA and Testing for DrugSensitivity

Constructs expressing shRNAs targeting SLFN12, or the control vector,were packaged into lentiviruses and delivered into HeLa cells by viraltransduction. Three SLFN12-targeting shRNAs were used, all of which wereobtained from the TRC (CloneIDs: TRCN0000152141 and TRCN0000153520).Infected cells were selected using 1 μg/ml puromycin for 3 days and thengrown in non-selective media for 3 more days. Cells were then platedinto 384-well assay plates and tested for drug sensitivity as describedabove. Knockdown of SLFN12 was validated by qPCR. Total RNA wasextracted using kit reagents (RNeasy Mini Kit (Qiagen #74104) andQIAschredder (Qiagen #79656)). cDNA was generated using kit reagents(SuperScript III First-Strand Synthesis System (Life Technologies#18080-051)). qPCR was performed for GAPDH and SLFN12 (Life TechnologiesHs00430118_ml) according to the manufacturer's recommendations. SLFN12expression was normalized to corresponding samples GAPDH ct-values.

Other Embodiments

From the foregoing description, it will be apparent that variations andmodifications may be made to the invention described herein to adopt itto various usages and conditions. Such embodiments are also within thescope of the following claims.

The recitation of a listing of elements in any definition of a variableherein includes definitions of that variable as any single element orcombination (or subcombination) of listed elements. The recitation of anembodiment herein includes that embodiment as any single embodiment orin combination with any other embodiments or portions thereof.

INCORPORATION BY REFERENCE

The ASCII text file submitted herewith via EFS-Web, entitled“167741_011202. txt” created on August [[TBD]], 2016, having a size of[[TBD]] bytes, is hereby incorporated by reference in its entirety.

All patents and publications mentioned in this specification are hereinincorporated by reference to the same extent as if each independentpatent and publication was specifically and individually indicated to beincorporated by reference. In particular, Lewis et al., “Compounds andCompositions for the Treatment of Cancer,” PCT/US2014/023263 (WO2014/164704) is incorporated by reference in its entirety.

What is claimed is:
 1. A method of reducing cancer cell proliferation,killing, or reducing the survival of a cancer cell selected asresponsive to a phosphodiesterase 3A (PDE3A) modulator, the methodcomprising contacting the cell with a PDE3A modulator that inducesinteraction between PDE3A and Schlafen 12 (SLFN12) proteins in the cell,wherein the cell was selected as having an increase in the level of aPDE3A and a SLFN12 polypeptide or polynucleotide relative to areference, thereby reducing cancer cell proliferation, killing, orreducing the survival of the cancer cell, wherein the PDE3A modulator is6-(4-(diethylamino)-3-nitrophenyl)-5-methyl-4,5-dihydropyridazin-3(2H)-one(DNMDP), or a pharmaceutically acceptable salt thereof.
 2. The method ofclaim 1, wherein the level of PDE3A or SLFN12 was detected by a methodselected from the group consisting of immunoblotting, mass spectrometry,and immunoprecipitation.
 3. The method of claim 1, wherein the level ofPDE3A or SLFN12 polynucleotide is detected by a method selected from thegroup consisting of quantitative PCR, Northern Blot, microarray, massspectrometry, and in situ hybridization.
 4. The method of claim 1,wherein the cancer cell is a melanoma, endometrium, lung,hematopoietic/lymphoid, ovarian, cervical, soft-tissue sarcoma,leiomyosarcoma, urinary tract, pancreas, thyroid, kidney, glioblastoma,or breast cancer cell.
 5. The method of claim 1, wherein the cancer cellis not a B-cell proliferative type cancer.
 6. The method of claim 1,wherein the cancer cell is not multiple myeloma.
 7. The method of claim1, wherein the PDE3A modulator reduces an activity of PDE3A.
 8. Themethod of claim 1, wherein the PDE3A modulator is administered orally.9. The method of claim 1, wherein the PDE3A modulator is administered byintravenous injection.
 10. The method of claim 1, wherein the cancer isa melanoma, endometrium, lung, hematopoietic/lymphoid, ovarian,cervical, soft-tissue sarcoma, leiomyosarcoma, urinary tract, pancreas,thyroid, kidney, glioblastoma, or breast cancer.